Antibody variants having modifications in the constant region

ABSTRACT

The present invention relates to positions in the constant region of antibodies, in particular the CH3 region of IgG4, which affect the strength of CH3-CH3 interactions. Mutations that either stabilize or destabilize this interaction are disclosed.

FIELD OF INVENTION

The present invention relates to modified antibodies that may be used intherapeutic applications. The invention also relates to methods forproducing the antibodies, pharmaceutical compositions comprising theantibodies and use thereof for different therapeutic applications.

BACKGROUND OF THE INVENTION

Native antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each light chain is comprised of a light chainvariable region (abbreviated herein as VL) and a light chain constantregion (abbreviated herein as CL). Each heavy chain is comprised of aheavy chain variable region (VH) and a heavy chain constant region (CH)consisting of three domain, CH1 , CH2 and CH3). CH1 and CH2 of the heavychain are separated from each other by the so-called hinge region. Thehinge region normally comprises one or more cysteine residues, which mayform disulphide bridges with the cysteine residues of the hinge regionof the other heavy chain in the antibody molecule.

Recently, antibodies have become a major focus area for therapeuticapplications, and many antibody drug products have been approved or arein the process of being approved for use as therapeutic drugs. Thedesired characteristics of therapeutic antibodies may vary according tothe specific condition which is to be treated. For some indications,only antigen binding is required, for instance where the therapeuticeffect of the antibody is to block interaction between the antigen andone or more specific molecules otherwise capable of binding to theantigen. For such indications, the use of Fab fragments, the onlyfunction of which is to bind antigen, may be preferred. For otherindications, further effects may also be required, such as for instancethe ability to induce complement activation and/or the ability to forinstance bind Fc receptors, protect from catabolism, recruit immunecells, etc. For such use, other parts of the antibody molecule, such asthe Fc region, may be required. Some full-length antibodies may exhibitagonistic effects (which may be considered to be undesirable) uponbinding to the target antigen, even though the antibody works as anantagonist when used as a Fab fragment. In some instances, this effectmay be attributed to “cross-linking” of the bivalent antibodies, whichin turn promotes target dimerization, which may lead to activation,especially when the target is a receptor. In the case of solubleantigens, dimerization may form undesirable immune complexes.

For some indications, monovalent antibodies may thus be preferable. Thepresently available Fab fragments show inferior pharmacokinetics due totheir small size resulting to filtration in the kidneys as well as theirinability to interact with the Brambell receptor FcRn (Junghans R P etal, Proc Natl Acad Sci USA 93(11), 5512-6 (1996)), therefore beingunstable in vivo and having very rapid clearance after administration.

There is thus a need for stable monovalent antibodies which can be usedas therapeutics.

Dimeric, monovalent antibodies (Fab/c), wherein the Fc region comprisestwo Fc polypeptides, have been described (WO200563816 to Genentech andParham P, J Immunol. 131(6), 2895-902 (1983).

Ig half-molecules, which have a dimeric configuration consisting of onlyone light chain and only one heavy chain, have been described as theresult of rare deletions in human and murine plasmacytomas. Studies onthe biochemical nature of these half-molecules showed that they consistof IgG1 molecules in which the heavy chain CH1, hinge and CH2 regionsappeared normal, whereas deletions were found in the CH3 region. Themutations appeared to be located in CH3 and the hinge peptide appearednormal (Hobbs, J R et al., Clin Exp Immunol 5, 199 (1969); Hobbs, J R,Br Med J 2, 67 (1971); Spiegelberg, H L et al., Blood 45, 305 (1975);Spiegelberg, H L et al., Biochemistry 14, 2157 (1975); Seligmann M E etal., Ann Immunol (Paris) 129C, 855-870 (1978); Gallango, M L et al.,Blut 48, 91 (1983)). It was also showed that this human IgG1half-molecule is rapidly catabolized (half-life in man was 4.3 days)and, in monomeric form, is unable to bind C1q or Fr receptors on humanlymphocytes, monocytes or neutrophils (Spiegelberg, H L. J Clin Invest56, 588 (1975)).

Murine IgA half-molecules which were generated by somatic mutation havealso been described (Mushinski, J F, J Immunol 106, 41 (1971);Mushinski, J F et al., J Immunol 117, 1668 (1976); Potter, M et al., JMol Biol 93, 537 (1964); Robinson, E A et al., J Biol Chem 249, 6605(1974); Zack, D J et :al., J Exp Med 154, 1554 (1981)). These moleculeswere shown to all contain deletions of the CH3 domain or mutations atthe CH2-CH3 boundary.

WO2007059782 (Genmab) describes human monovalent antibodies comprising alight chain and a heavy chain, wherein

a) said light chain comprises the amino acid sequence of the variable(VL) region of a selected antigen specific antibody and the amino acidsequence of the constant (CL) region of an Ig, and wherein, in case ofan IgG1 subtype, the amino sequence of the constant (CL) region has beenmodified so that it does not contain any amino acids capable ofparticipating in the formation of disulfide bonds or covalent bonds withother peptides comprising an identical amino acid sequence of theconstant (CL) region of the Ig, in the presence of polyclonal human IgGor when administered to an animal or human being, and

b) said heavy chain comprises the amino acid sequence of the variable(VH) region of said selected antigen specific antibody and the aminoacid sequence of the constant (CH) region of human Ig, wherein the aminoacid sequence of the constant (CH) region has been modified so that thehinge region and, as required by the Ig subtype, other regions of the CHregion, such as the CH3 region, does not contain any amino acid residueswhich participate in the formation of disulphide bonds or covalent orstable non-covalent inter-heavy chain bonds with other peptidescomprising an identical amino acid sequence of the constant (CN) regionof the human Ig, in the presence of polyclonal human IgG or whenadministered to an animal or human being.

As shown in WO2007059782, these monovalent antibodies have a morefavorable in vivo half-life than Fab fragments. WO2008145140 describesvariants of these monovalent antibodies wherein intermolecular CH3-CH3interactions are destabilized. The present application describesalternative and improved variants of the monovalent antibodies disclosedin WO2007059782 and WO2008145140. These variants remain monovalent evenunder conditions that favor intermolecular CH3-CH3 interactions.

Human IgG4 molecules exist in various molecular forms which differ bythe absence or presence of inter-heavy chain disulphide bonds located inthe hinge region. Thus IgG4 molecules exist in which two, one or nointer-heavy chain disulphide bonds have been formed (Schuurman, J. etal., Mol immunol 38, 1 (2001)). Under physiological conditions, thesemolecular forms of IgG4 may be in equilibrium with each other. HumanIgG4s exist as tetramers in solution consisting of two Ig heavy and twolight chains, as common for immunoglobulin G molecules, irrespective ofthe absence or presence of these interchain disulphide bonds (Schuurman2001 supra; Gregory, L. et al. Mol Immuno) 24, 321 (1987)). Only upondenaturation under non-reducing conditions, the two non-covalentlyassociated half-molecules dissociate as demonstrated bysize-determination analysis such as SDS-PAGE (Schuurman, J. et al. MolImmunol) 38, 1 (2001); Deng, L. et al. Biotechnol Appl Biochem 40, 261(2004)). It has been shown that mutation of the residues of the hingeregion which are involved in inter-chain disulphide bond formation ordeletion of the hinge region lead to creation of a homogeneous pool ofIgG4 molecules in solution, which pool consists of tetrameric moleculesconsisting of two light chains and two heavy chains (Schuurman, J. etal. Mol Immunol 38. 1 (2001); Horgan, C. et al. J Immunol 150, 5400(1993)). The IgG4 hinge-deleted and mutated antibodies also demonstratedan improved capability of antigen crosslinking when compared to nativeIgG₄ molecules (Horgan, C. (1993) supra).

It has been shown that administration of two recombinant monoclonal IgG4antibodies having different antigen-binding specificities to a mouseleads to in vivo formation of bispecific antibodies. The phenomenon canbe reproduced in vitro by incubating IgG4 antibodies with cells or underreducing conditions. It has been shown that IgG4 antibodies havingdifferent antigen-binding specificities engage in Fab arm exchange whichis stochastic and in which all IgG4 molecules seem to participate. Thus,IgG4 antibodies form bispecific antibodies without concomitant formationof aggregates.

IgG4 antibodies therefore have unusual properties which are undesirablein vivo; IgG4 antibodies are unstable, dynamic, molecules which engagein Fab arm exchange. An administered therapeutic IgG4 antibody mayexchange with endogenous IgG4 antibodies with undesired specificities.The random nature of this process introduces unpredictability which ishighly undesirable for human immunotherapy.

In one aspect, the present invention relates to stabilized forms of IgG4antibodies that have a reduced ability to undergo Fab-arm exchange.Stabilized forms of IgG4 have previously been described in WO2008145142(Genmab). It has now surprisingly been found that specific alternativesubstitutions in human IgG4 can prevent Fab arm exchange, and thusstabilize IgG4.

In summary, the present invention relates to positions in the constantregion of antibodies, in particular the CH3 region of IgG4, which affectthe strength of CH3-CH3 interactions. Mutations that either stabilize ordestabilize this interaction are disclosed herein.

When introduced in the monovalent antibody context described inWO2007059782, the destabilizing mutations contribute to keeping theantibodies monovalent even under conditions that favor intermolecularCH3-CH3 interactions. When introduced in the IgG4 context, thestabilizing mutations contribute to preventing undesired Fab armexchange.

SUMMARY OF THE INVENTION

In a first main aspect, the invention relates to a monovalent antibody,which comprises

(i) a variable region of a selected antigen specific antibody or anantigen binding part of the said region, and

(ii) a CH region of an immunoglobulin or a fragment thereof comprisingthe CH2 and CH3 regions, wherein the CH region or fragment thereof hasbeen modified such that the region corresponding to the hinge regionand, if the immunoglobulin is not an IgG4 subtype, other regions of theCH region, such as the CH3 region, do not comprise any amino acidresidues which are capable of forming disulfide bonds with an identicalCH region or other covalent or stable non-covalent inter-heavy chainbonds with an identical CH region in the presence of polyclonal humanIgG,

wherein the antibody is of the IgG4 type and the constant region of theheavy chain has been modified so that one or more of the following aminoacid substitutions have been made relative to the sequence set forth inSEQ ID NO:4: Tyr (Y) in position 217 has been replaced by Arg (R), Leu(L) in position 219 has been replaced by Asn (N) or Gln (Q), Gln (E) inposition 225 has been replaced by Thr (T), Val (V) or IIe (I), Ser (S)in position 232 has been replaced by Arg (R) or Lys (K), Thr (T) inposition 234 has been replaced by Arg (R), Lys (K) or Asn (N), Leu (L)in position 236 has been replaced by Ser (S) or Thr (T), Lys (K) inposition 238 has been replaced by Arg (R), Asp (D) in position 267 hasbeen replaced by Thr (T) or Ser (S), Phe (F) in position 273 has beenreplaced by Arg (R), Gln (Q), Lys (K) or Tyr (Y), Tyr (Y) in position275 has been replaced by Gln (Q), Lys (K) or Phe (F), Arg (R) inposition 277 has been replaced by Glu (E), Thr (T) in position 279 hasbeen replaced by Asp (D), Val (V) and Asn (N),

or the antibody is of another IgG type and the constant region of theheavy chain has been modified so that one or more of the same amino-acidsubstitutions have been made at the positions that correspond to thebefore-mentioned positions for IgG4.

As explained above, mutations at the above specified positions disfavorintermolecular CH3-CH3 interactions. Thus, monovalent antibodiescarrying these mutations are less likely to dimerize throughnon-covalent interactions. This may be an advantage for therapeuticapplications wherein such dimerization is highly undesired. Furthermore,a reduced tendency of the monovalent antibodies to associatenon-covalently through the CH3 regions may make pharmaceuticalcompositions comprising such antibodies more stable and homogenous thanpharmaceutical compositions of monovalent antibodies that do notcomprise the above-specified mutations.

Thus, in another aspect, the invention relates to a pharmaceuticalcomposition comprising the monovalent antibody according the inventionas defined herein.

In a further aspect, the invention relates to a method of treating adisease or disorder as described herein, wherein said method comprisesadministering to a subject in need of such treatment a therapeuticallyeffective amount of a monovalent antibody according to the invention.

In a further aspect, the invention relates to a stabilized IgG4 antibodyfor use as a medicament, comprising a heavy chain and a light chain,wherein said heavy chain comprises a human IgG4 constant region havingthe sequence set forth in SEQ ID NO:2, wherein Lys (K) in position 250has been replaced by Gln (Q) or Glu (E) and wherein the antibodyoptionally comprises one or more further substitutions, deletions and/orinsertions in the constant region as set forth in SEQ ID NO:2.

As explained above, and shown herein below in the Examples, themutations at position 250 stabilize the IgG4 molecule and preventundesired Fab arm exchange.

DESCRIPTION OF FIGURES

FIG. 1: Percentage of molecules present as monomers for each HG mutanttested using non-covalent nano-electrospray mass spectrometry. HG mutantsamples were prepared in aqueous 50 mM ammonium acetate solutions at aconcentration of 1 μM.

FIG. 2: NativePAGE™ Novex® Bis-Tris gel electrophoresis of CH3 mutantscompared to 2F8-HG (WT) and R277K HG mutant control.

FIG. 3: The binding of 2F8-HG and CH3 variants 2F8-HG-T234A and2F8-HG-L236V was tested in EGFR ELISA in the presence and absence ofpolyclonal human IgG.

FIG. 4: The binding of 2F8-HG and CH3 variants 2F8-HG-L236A and2F8-HG-Y275A was tested in EGFR ELISA in the presence and absence ofpolyclonal human IgG.

FIG. 5: Dose-response curves showing the inhibition of EGF-induced EGFrphosphorylation in A431 cells by anti-EGFr 2F8-HG (WT) and CH3 mutantsthereof.

FIG. 6: Percentage molecules present as monomers at different molarconcentrations of CH3 mutants compared to 2F8-HG (WT) and 1277K.

FIG. 7: Relative interaction strength (KD) of CH3 mutants compared to2F8-HG (WT).

FIG. 8: The binding of 2F8-HG and deglycosylation variants 2F8-HG-GSTand 2F8-HG-NSE was tested in EGFR ELISA in the presence and absence ofpolyclonal human IgG.

FIG. 9: Percentage of molecules present as monomers for each HG mutantmeasured using non-covalent nano-electrospray mass spectrometry. HGmutant samples were prepared in aqueous 50 mM ammonium acetate solutionsat a concentration of 1 μM.

FIG. 10: Dose-response curves showing the inhibition of EGF-induced EGFrphosphorylation in A431 cells by anti-EGFr 2F8-HG (WT) andnon-glycosylation mutants thereof.

FIG. 11: Clearance (expressed as D/AUC) of non-glycosylation mutants2F8-HG-GST and 2F8-HG-NSE compared to 28-HG (WT) and 2F8-IgG4.

FIG. 12: Schematic representation of constructs for IgG1 and IgG4containing mutations in the core hinge and/or CH3 domain (residues arenumbered according to EU numbering, see table Example 16).

FIG. 13: Fab arm exchange of IgG1 and IgG4 hinge region or CH3 domainmutants (residues are numbered according to EU numbering, see tableExample 16).

FIG. 14: Binding of hingeless IgG4 antibody 2F8-HG and CH3 variants2F8-HG-F405L, 2F8-HG-F405A, 2F8-HG-R409A and 2F8-HG-R409K to EGFr(residues are numbered according to EU numbering, see table Example 16).Binding was tested in an EGFR ELISA in the presence and absence ofpolyclonal human IgG (IVIG).

FIG. 15: Sequence alignment of anti-EGFr antibody 2F8 in an IgG1, (IgG4and (partial) IgG3 backbone. Amino acid numbering according to Kabat andaccording to the EU-index are depicted (both described in Kabat et al.,Sequences of Proteins of immunological Interest. 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)).

FIG. 16: Fab-arm exchange of CH3 domain mutants of human IgG4antibodies. Mixtures of two recombinant human IgG4 antibodies (IgG4-CD20and IgG4-EGFr) and CH3 domain mutants thereof were incubated with 0.5 mMGSH at 37° C. The formation of bispecific antibodies through Fab armexchange was followed over time and measured in a sandwich ELISA. Thebispecific activity of IgG4 at 24 hrs was set as 100%.

FIG. 17: Relative interaction strength (K_(D)) of CH3 mutants comparedto his-CH2-CH3(G4) (WT).

FIG. 18: Correlation between the CH3-CH3 interaction strength (K_(D))and the bispecific activity. The bispecific activity of IgG4 at 24 hrswas set as 100% (open circle).

DETAILED DESCRIPTION OF THE SEQUENCE LISTINGS

SEQ ID No:1: The nucleic acid sequence of the wildtype CH region ofhuman IgG4

SEQ ID No:2: The amino acid sequence of the wildtype CH region of humanIgG4.

Sequences in italics represent the CH1 region, highlighted sequencesrepresent the hinge region, regular sequences represent the CH2 regionand underlined sequences represent the CH3 region.

SEQ ID No: 3: The nucleic acid sequence of the CH region et human IgG4(SEQ ID No: 1) mutated in positions 714 and 722

SEQ ID No: 4: The amino acid sequence of the hingeless CH region of ahuman IgG4. Underlined sequences represent the CH3 region.

SEQ) ID No: 5: The amino acid sequence of the lambda chain constanthuman (accession number S25751)

SEQ ID No: 6: The amino acid sequence of the lambda chain constant human(accession number P01834)

SEQ ID No: 7: The amino acid sequence of IgG1 constant region (accessionnumber P01857). Sequences in italics represent the CH1 region,highlighted sequences represent the hinge region, regular sequencesrepresent the CH2 region and underlined sequences represent the CH3region

SEQ ID No: 8: The amino acid sequence of the IgG2 constant region(accession number P01859). Sequences in italics represent the CH1region, highlighted sequences represent the hinge region, regularsequences represent the CH2 region and underlined sequences representthe CH3 region

SEQ ID No: 9: The amino acid sequence of the IgG3 constant region(accession number A23511). Sequences in italics represent the CH1region, highlighted sequences represent the hinge region, regularsequences represent the CH2 region and underlined sequences representthe CH3 region

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “antibody” as referred to herein includes whole antibodymolecules, antigen binding fragments, monovalent antibodies, and singlechains thereof. Antibody molecules belong to a family of plasma proteinscalled immunoglobulins, whose basic building block, the immunoglobulinfold or domain, is used in various forms in many molecules of the immunesystem and other biological recognition systems. Native antibodies andimmunoglobulins are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries between the heavy chains of different immunoglobulin isotypes.Each heavy and light chain may also have regularly spaced intrachaindisulfide bridges. Each light chain is comprised of a light chainvariable region (abbreviated herein as VL) and a light chain constantregion (abbreviated herein as CL). Each heavy chain is comprised of aheavy chain variable region (VH) and a heavy chain constant region (CH)consisting of three domains, CH-1, CH2 and CH3, and the hinge region).The three CH domains and the hinge region have been indicated for IgG1IgG2, IgG3 and IgG4 in SEQ) ID NO: 7, 8, 9 and 2, respectively (seebelow). The constant domain of the light chain is aligned with the firstconstant domain (CH1) of the heavy chain, and the light chain variabledomain is aligned with the variable domain of the heavy chain formingwhat is known as the “Fab fragment”. CH1 and CH2 of the heavy chain areseparated form each other by the so-called hinge region, which allowsthe Fab “arms” of the antibody molecule to swing to some degree. Thehinge region normally comprises one or more cysteine residues, which arecapable of forming disulphide bridges with the cysteine residues of thehinge region of the other heavy chain in the antibody molecule.

The variable regions of the heavy and light chains contain a bindingdomain that interacts with an antigen. The constant regions of theantibodies may mediate the binding of the immunoglobulin to host tissuesor factors, including various cells of the immune system (for instanceeffector cells) and the first component (C1q) of the classicalcomplement system

Depending on the amino acid sequences of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are at least five (5) major classes of immunoglobulins: IgA, IgD,IgE, IgG and IgM, and several of these may be further divided intosubclasses (isotypes), for instance IgG1. IgG2, IgG3 and IgG4.; IgA1 andIgA2. The genes for the heavy chains constant domains that correspond tothe different classes of immunoglobulins are called alpha (α), delta(õ), epsilon (ε), gamma (γ) and mu (μ), respectively. Immunoglobulinsubclasses are encoded by different genes such as γ1, γ2, γ3 and γ4. Thegenes for the light chains of antibodies are assigned to one of twoclearly distinct types, called kappa (κ) and lambda (λ), based on theamino sequences of their constant domain. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known. Distinct allotypes of immunoglobulins exist within thehuman population such as G1m(a), G1m(x), G1m(f) and G1m(z) for IgG1heavy chain and Km1 , Km1,2 and Km3 for the kappa light chain. Theseallotypes differ at distinct amino acids in their region encoding theconstant regions.

The term antibody also encompasses “derivatives” of antibodies, whereinone or more of the amino acid residues have been derivatised, forinstance by acylation or glycosylation, without significantly affectingor altering the binding characteristics of the antibody containing theamino acid sequences.

In addition, the term antibody covers variants, e.g. variants whereinthe in vivo half-life of the antibodies has been improved by modifyingthe salvage receptor epitope of the Ig constant domain or an Ig-likeconstant domain such that the molecule does not comprise an intact CH2domain or an intact Ig Fc region, cf. U.S. Pat. No. 6,121,022 and U.S.Pat. No. 6,194,551. The in vivo half-life may be furthermore increasedby making mutations in the Fc region, for instance by substitutingthreonine for leucine at the position corresponding to position 252 ofan intact antibody molecule, threonine for serine at the positioncorresponding to position 254 of an intact antibody molecule, orthreonine for phenylalanine at the position corresponding to position256 of an intact antibody molecule, cf. U.S. Pat. No. 6,277,375.

Furthermore, antibodies, and particularly Fab or other fragments, may bepegylated to increase the half-life. This can be carried out bypegylation reactions known in the art, as described, for example, inFocus on Growth Factors 3, 4-10 (1992), EP 154 316 and EP 401 384.

The term “antibody half-molecule” is used herein to mean an antibodymolecule as described above, but comprising no more than one light chainand no more than one heavy chain, and which exists in water solutions asa heterodimer of said single light and single heavy chain. Such antibodyis by nature monovalent as only one antigen-binding portion is present.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (for instance mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in viva).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR1 or CDR2 sequences derived from thegermline of another mammalian species, such as a mouse, or the CDR3region derived from an antibody from another species, such as mouse,have been grafted onto human framework sequences. Human monoclonalantibodies directed may be generated using transgenic ortranschromosomal mice carrying parts of the human immune system ratherthan the mouse system. Such transgenic and transchromosomic mice includemice referred to herein as HuMAb mice and KM mice, respectively, and arecollectively referred to herein as “transgenic mice”.

The HuMAb mouse contains a human immunoglobulin gene miniloci thatencodes unrearranged human heavy (μ and γ) and κ light chainimmunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (Lonberg, N. et al., Nature368, 856-859 (1994)). Accordingly, the mice exhibit reduced expressionof mouse IgM or κ and in response to immunization, the introduced humanheavy and light chain transgenes, undergo class switching and somaticmutation to generate high affinity human IgG,κ monoclonal antibodies(Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. Handbook ofExperimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Hussar,D., Intern. Rev. Immunol. Vol. 13 65-93 (1995) and Harding, F. andLonberg, N. Ann. N.Y. Acad. Sci 764 536-546 (1995)). The preparation ofHuMAb mice is described in detail in Taylor, L. et al., Nucleic AcidsResearch 20, 6287-6295 (1992), Chen, J. et al., International immunology5, 647-656 (1993), Tuaillon et al., J. Immunol. 152. 2912-2920 (1994),Taylor, L. et al., International Immunology 6, 579-591 (1994), Fishwild,D. et al., Nature Biotechnology 14, 845-851 (1996). See also U.S. Pat.No. 5,545,806, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,625,126, U.S.Pat. No. 5,633,425, U.S. Pat. No. 5,789,650, U.S. Pat. No. 5,877,397,U.S. Pat. No. 5,661,016, U.S. Pat. No. 5,814,318, U.S. Pat. No.5,874,299, U.S. Pat. No. 5,770,429, U.S. Pat. No. 5,545,807, WO98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO01/09137.

The HCo7 mice have a JKD disruption in their endogenous light chain(kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)),a CMD disruption in their endogenous heavy chain genes (as described inExample 1 of WO 01/14424), a KCo5 human kappa light chain transgene (asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)),and a HCo7 human heavy chain transgene (as described in U.S. Pat. No.5,770,429).

The HCo12 mice have a JKD disruption in their endogenous light chain(kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)),a CMD disruption in their endogenous heavy chain genes (as described inExample 1 of WO 01/14424), a KCo5 human kappa light chain transgene (asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)),and a HCo12 human heavy chain transgene (as described in Example 2 of WO01/14424).

In the KM mouse strain, the endogenous mouse kappa light chain gene hasbeen homozygously disrupted as described in Chen et al., EMBO J. 12,811-820 (1993) and the endogenous mouse heavy chain gene has beenhomozygously disrupted as described in Example 1 of WO 01/09187. Thismouse strain carries a human kappa light chain transgene, KCo5, asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996).This mouse strain also carries a human heavy chain transchromosomecomposed of chromosome 14 fragment hCF (SC20) as described in WO02/43478.

Splenocytes from these transgenic mice may be used to generatehybridomas that secrete human monoclonal stabilized IgG4 antibodiesaccording to well known techniques. Such transgenic non-human animals,non-human animals comprising an operable nucleic acid sequence codingfor expression of antibody used in the invention, non-human animalsstably transfected with one or more target-encoding nucleic acidsequences, and the like, are additional features of the presentinvention. The term “K_(D)” (M), as used herein, refers to thedissociation equilibrium constant of a particular antibody-antigeninteraction.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.Accordingly, the term “human monoclonal antibody” refers to antibodiesdisplaying a single binding specificity which have variable and constantregions derived from human germline immunoglobulin sequences.

The term “monovalent antibody” means in the present context that anantibody molecule is capable of binding a single molecule of theantigen, and thus is not able of antigen crosslinking.

As used herein, “specific binding”′ refers to the binding of anantibody, or antigen-binding fragment thereof, to a predeterminedantigen. Typically, the antibody binds with an affinity corresponding toa K_(D) of about 10⁻⁷ M or less, such as about 10⁻⁹ M or less, such asabout 10⁻⁹ M or less, about 10⁻¹⁰ M or less, or about 10⁻¹¹ M or evenless, when measured for instance using sulfon plasmon resonance onBlAcore or as apparent affinities based on IC₅₀ values in FACS or ELISA,and binds to the predetermined antigen with an affinity corresponding toa K_(C) that is at least ten-fold lower, such as at least 100 foldlower, for instance at least 1000 fold lower, such as at least 10,000fold lower, for instance at least 100,000 told lower than its affinityfor binding to a non-specific antigen (e.g., BSA, casein) other than thepredetermined antigen or a closely-related antigen. The amount withwhich the affinity is lower is dependent on the K_(D) of the antigenbinding peptide, so that when the K_(D) of the antigen binding peptideis very low (that is, the antigen binding peptide is highly specific),then the amount with which the affinity for the antigen is lower thanthe affinity for a non-specific antigen may be at least 10,000 fold.

The terms “transgenic, non-human animal” refers to a non-human animalhaving a genome comprising one or more human heavy and/or light chaintransgenes or transchromosomes (either integrated or non-integrated intothe animal's natural genomic DNA) and which is capable of expressinghuman antibodies. For example, a transgenic mouse can have a human lightchain transgene and either a human heavy chain transgene or human heavychain transchromosome, such that the mouse produces human antibodieswhen immunized with an antigen and/or cells expressing an antigen. Thehuman heavy chain transgene can be integrated into the chromosomal DNAof the mouse, as is the case for transgenic, for instance HuMAb mice,such as HCo7 or HCo12 mice, or the human heavy chain transgene can bemaintained extrachromosomally, as is the case for transchromosomal KMmice as described in WO 02/43478. Such transgenic and transchromosomalmice are capable of producing multiple classes and isotypes ofmonovalent antibodies to a given antigen (for instance IgM, IgG, IgAand/or IgE) by undergoing V-D-J recombination and isotype switching.

The term “acceptor site for N-linked glycosylation” refers to a site ona polypeptide which is susceptible of becoming glycosylated on an Asnresidue. The typical consensus site for this type of glycosylation isAsn-X-Ser/Thr, wherein X can be any amino acid, except for Pro.

As explained above, the characteristic IgG structure in which twoheavy-light chain heterodimers are linked is maintained by theinter-heavy chain disulphide bridges of the hinge region and thenon-covalent interactions of the CH3 domains.

It has been shown in WO2007059782 that removal of the hinge region inIgG4 results in the formation of monovalent antibodies in which thelinkage between the two heavy-light chain heterodimers is lost ordiminished. Consequently, changes in hinge region disulphide bridges ofother IgG subclasses alone or in combination with mutations in the CH3domain interactions may result in the formation of monovalent antibodiesfor these other subclasses as well. It is well within the capability ofthe skilled artisan to use the intimate knowledge of structure of Igsubclasses, and the knowledge provided in the present invention, toselect and to modify selected amino acids to prevent light chaininteractions.

In a first main aspect, the invention relates to a monovalent antibody,which comprises

(i) a variable region of a selected antigen specific antibody or anantigen binding part of the said region, and

(ii) a CH region of an immunoglobulin or a fragment thereof comprisingthe CH2 and CH3 regions, wherein the CH region or fragment thereof hasbeen modified such that the region corresponding to the hinge regionand, if the immunoalobulin is not an IgG4 subtype, other regions of theCH region, such as the CH3 region, do not comprise any amino acidresidues which are capable of forming disulfide bonds with an identicalCH region or other covalent or stable non-covalent inter-heavy chainbonds with an identical CH region in the presence of polyclonal humanIgG.

wherein the antibody is of the IgG4 type and the constant region of theheavy chain has been modified so that one or more of the following aminoacid substitutions have been made relative the sequence set forth in SEQID NO: 4: Tyr (Y) in position 217 has been replaced by Arg (R), Leu (L)in position 219 has been replaced by Asn (N) or Gln (Q), Glu (E) inposition 225 has been replaced by Thr (T), Val (V) or IIe (I), Ser (S)in position 232 has been replaced by Arg (A) or Lys (K), Thr (T) inposition 234 has been replaced by Arg (R), Lys (K) or Asn (N), Leu (L)in position 236 has been replaced by Ser (S) or Thr (T), Lys (K) inposition 238 has been replaced by Arg (R), Asp (D) in position 267 hasbeen replaced by Thr (T) or Ser (S), Phe (F) in position 273 has beenreplaced by Arg (R), Gln (Q), Lys (K) or Tyr (Y), Tyr (Y) in position275 has been replaced by Gln (Q), Lys (K) or Phe (F), Arg (R) inposition 277 has been replaced by Glu (E), Thr (T) in position 279 hasbeen replaced by Asp (D), Val (V) and Asn (N).

or the antibody is of another IgG type and the constant region of theheavy chain has been modified so that one or more of the same amino-acidsubstitutions have been made at the positions that correspond to thebefore-mentioned positions for lgG4. See e.g. SEQ ID NO: 7, 8 and 9 forthe corresponding positions in other isotypes.

In one embodiment, the monovalent antibody comprises

(i) a variable region of a selected antigen specific antibody or anantigen binding part of the said region, and

(ii) a CH region of an immunoglobulin or a fragment thereof comprisingthe CH2 and CH3 regions, wherein the CH region or fragment thereof hasbeen modified such that the region corresponding to the hinge regionand, if the immunoglobulin is not an IgG4 subtype, other regions of theCH region, such as the CH3 region, do not comprise any amino acidresidues which are capable of forming disulfide bonds with an identicalCH region or other covalent or stable non-covalent inter-heavy chainbonds with an identical CH region in the presence of polyclonal humanIgG.

wherein the antibody is of the IgG4 type and the constant region of theheavy chain has been modified so that one or more of the following aminoacid substitutions have been made relative the sequence set forth in SEQID NO: 4: Glu (E) in position 225 has been replaced by Val (V), Ser (S)in position 232 has been replaced by Arg (R), Leu (L) in position 236has been replaced by Ser (S) or Thr (T), Asp (D) in position 267 hasbeen replaced by Thr (T) or Ser (S), Phe (F) in position 273 has beenreplaced by Arg (R), Gln (Q) or Tyr (Y), Tyr (Y) in position 275 hasbeen replaced by Gln (Q) or Lys (K).

In another embodiment, the antibody is of the IgG4 type and the constantregion of the heavy chain has been modified so that one or more of thefollowing combinations of amino acid substitutions have been maderelative the sequence set forth in SEQ ID NO: 4:

-   Asp (D) in position 267 has been replaced by Ser (S) and Tyr (Y) in    position 275 has been replaced by Gln (Q) or Lys (K), Arg (R),-   Asp (D) in position 267 has been replaced by Thr (T) and Tyr (Y) in    position 275 has been replaced by Gln (Q) or Lys (K), Arg (R),

or the antibody is of another IgG type and the constant region of theheavy chain has been modified so that the same combinations ofamino-acid substitutions have been made at the positions that correspondto the before-mentioned positions for IgG4.

Typically, the variable region and the C_(H) region of the monovalentantibody are connected to each other via peptide bonds and are producedfrom a single open reading frame. Without being bound to any theory, itis believed that the monovalent antibodies according to the inventionare capable of binding to the FcRn. Such binding may be determined byuse of methods for determining binding as it is known in the art, forinstance by use of ELSA assays. The binding of a monovalent antibody ofthe invention to FcRn may for instance be compared to the binding of aF(ab′)₂ fragment, which F(ab′)₂ fragment has a VH region and a VLregion, which are identical to the VH region and the VL region of themonovalent antibody of the invention, to FcRn in the same assay. In oneembodiment, the binding of a monovalent antibody of the invention toFcRn is more than 10 times stronger than the binding of the F(ab′)₂fragment to FcRn.

In one embodiment, the antibody (further) comprises a CH1 region.

In another embodiment, the monovalent antibody consists of said variableregion and said CH region.

In another embodiment, the variable region is a VH region. In a furtherembodiment, the variable region is a VL region. In an even furtherembodiment the antibody does not comprise a CL region.

In an important embodiment, the monovalent antibody of the inventioncomprises a heavy chain and a light chain, wherein the heavy chaincomprises

-   -   (i) a VH region of a selected antigen specific antibody or an        antigen binding part of the said region, and    -   (ii) a CH region as defined above,        and the light chain comprises    -   (i) a VL region of a selected antigen specific antibody or an        antigen binding part of the said region, and    -   (ii) a CL region which, in case of an IgG1 subtype, has been        modified such that the CL region does not contain any amino        acids, which are capable of forming disulfide bonds with an        identical CL region or other covalent bonds with an identical CL        region in the presence of polyclonal human IgG.

Typically, the light chain and the heavy chain of the monovalentantibody defined above are connected to each other via one or moredisulfide bonds. It is evident that for such disulphide bonds, neitherof the binding partners in the disulphide, bond is present in the regioncorresponding to the hinge region. In one embodiment however the lightchain and the heavy chain of the monovalent antibody are connected toeach other via one or more amide bonds.

Furthermore, typically, the VL region and the CL region of the lightchain are connected to each other via peptide bonds and produced from asingle open reading frame.

In one embodiment, the VH and VL region of an antibody molecule of theinvention are derived from the same antigen specific antibody.

According to the invention, the, sequence of the CL region of the lightchain of the antibody molecule may be derived from the sequence of CLregion of an immunoglobulin. In one embodiment, the CL region is theconstant region of the kappa light chain of human IgG. In oneembodiment, the CL region comprises the amino acid sequence of SEQ IDNo: 2. In one embodiment, the CL region is the constant region of thelambda light chain of human IgG. In one embodiment, the CL regioncomprises the amino acid sequence of SEQ ID No: 4.

In one embodiment, the monovalent antibody of the invention is an IgC1,IgG2, IgG3, IgG4, IgA or IgD antibody, such as an IgG1, IgG2 or IgG4antibody. In a further embodiment, the monovalent antibody is a humanantibody.

A monovalent antibody of the present invention may also be a variant ofany of the above isotypes. For example, a variant IgG4 antibody may bean antibody that differs from a IgG4 antibody by one or more suitableamino acid residue alterations, that is substitutions, deletions,insertions, or terminal sequence additions, for instance in the constantdomain, and/or the variable regions (or any one or more CDRs thereof) ina single variant antibody. Typically, amino acid sequence alterations,desirably do not substantially change the structural characteristics ofthe parent sequence (e.g., a replacement amino acid should not tend todisrupt secondary structure that characterizes the function of theparent sequence), but which may be associated with advantageousproperties, such as changing the functional or pharmacokineticproperties of the antibodies, for example increasing the half-life,altering the immunogenicity, providing a site for covalent ornon-covalent binding to another molecule, reducing susceptibility toproteolysis or reducing susceptibility to oxidation. Examples ofvariants include variants which have a modification of the CH3 region,such as a substitution or deletion at any one or more of the positions225, 234, 236, 238, 273 or 275 of SEQ ID NO: 4 or the correspondingresidues in non-IgG4 isotypes. Modifications at these positions may e.g.further reduce intermolecular interactions between hinge-modifiedantibodies of the invention. Other examples include variants which havea modification of the constant region, such as a substitution ordeletion, at any one or more of the positions 118, 120, 122, 124, 175,248, 298, 302 of SEQ ID NO: 4 or the corresponding residues in non-IgG4isotypes. Modifications at these positions may e.g. increase thehalf-life of hinge-modified antibodies of the invention.

In one embodiment, the monovalent antibody of the invention comprisesthe CH3 region as set as set forth in SEQ ID NO: 7, but wherein the CH3region has been modified so that one or more of the following amino acidsubstitutions have been made: Arg (R) in position 238 has been replacedby Gln (Q); Asp (D) in position 239 has been replaced by Glu (E); Thr(T) in position 249 has been replaced by Ala (A); Leu (L) in position251 has been replaced by Ala (A); Lau (L) in position 251 has beenreplaced by Val (V); Phe (F) in position 288 has been replaced by Ala(A); Phe (F) in position 288 has been replaced by Leu (L); Tyr (Y) inposition 290 has been replaced by Ala (A); Lys (K) in position 292 hasbeen replaced by Arg (R); Lys (K) in position 292 has been replaced byAla (A); Gln (Q) in position 302 has been replaced by Glu (F); and Pro(P) in position 328 has been replaced by Leu (L).

In a further embodiment hereof, one or more of the following amino acidsubstitutions have been made: Arg (R) in position 238 has been replacedby Gln (Q); Asp (D) in position 239 has beer replaced by Gln (E); Lys(K) in position 292 has been replaced by Arg (R); Gln (Q) in position302 has been replaced by Glu (E); and Pro (P) in position 328 has beenreplaced by Leu (L). In an even further embodiment:

-   (i) Arg (R) in position 238 has been replaced by Gln (Q),-   (ii) Arg (R) in position 238 has been replaced by Gln (Q), and    Pro (P) in position 328 has been replaced by Leu (L), or-   (iii) all five substitutions as defined above have been made.

In another further embodiment hereof, the monovalent antibody furthercomprises the CH1 and/or CH2 regions as set forth in SEQ ID NO: 7, withthe proviso that the CH2 region has been modified so that it does notcomprise any acceptor sites for N-linked glycosylation.

In one embodiment, the monovalent antibody of the invention comprisesthe kappa CL region having the amino acid sequence as sot forth in SEQID NO: 6, but wherein the sequence has been modified so that theterminal cysteine residue in position 106 has been replaced with anotheramino acid residue or has been deleted.

In another embodiment, the monovalent antibody of the inventioncomprises the lambda CL region having the amino acid sequence as setforth in SEQ ID NO: 5, but wherein the sequence has been modified sothat the cysteine residue in position 104 has been replaced with anotheramino acid residue or has been deleted.

In a further embodiment, the monovalent antibody of the inventioncomprises the CH1 region as set forth in SEQ ID NO: 7, but wherein theCH1 region has been modified so that Ser (S) in position 14 has beenreplaced by a cysteine residue.

In a different embodiment, the monovalent antibody of the inventioncomprises the CH3 region as set forth in SEQ ID NO: 8, but wherein theCH3 region has been modified so that one or more of the of the followingamino acid substitutions have been made: Arg (R) in position 234 hasbeen replaced by Gln (Q); Thr (T) in position 245 has been replaced byAla (A); Leo (L) in position 247 has been replaced by Ala (A); Leu (L)in position 247 has been replaced by Val (V); Met (M) in position 276has been replaced by Val (V); Phe (F) in position 284 has been replacedby Ala (A); Phe (F) in position 284 has been replaced by Leu (L); Tyr(Y) in position 286 has been replaced by Ala (A); Lys (K) in position288 has been replaced by Arg (R); Lys (K) in position 288 has beenreplaced by Ala (A); Gln (Q) in position 298 has been replaced by Glu(E); and Pro (P) in position 324 has been replaced by Leu (L).

In a further embodiment hereof, one or more of the of the followingamino acid substitutions have been made: Arg (R) in position 234 hasbeen replaced by Gln (Q); Met (M) in position 276 has been replaced byVal (V); Lys (K) in position 288 has been replaced by Arg (R); Gln (Q)in position 298 has been replaced by Glu (E); and Pro (P) in position324 has been replaced by Leu (L). In an even further embodiment:

-   (i) Arg (R) in position 234 has been replaced by Gln (Q);-   (ii) Arg (R) in position 234 has been replaced by Gln (Q); and    Pro (P) in position 324 has been replaced by Leu (L); or-   (iii) all five substitutions as defined above have been made.

In another further embodiment hereof, the monovalent antibody furthercomprises the CH1 and/or CH2 regions as set forth in SEQ ID NO: 8, withthe proviso that the CH2 region has been modified so that it does notcomprise any acceptor sites for N-linked glycosylation.

In a further different embodiment, the monovalent antibody of theinvention comprises the CH3 region as set forth in SEQ ID NO: 9, butwherein the CH3 region has been modified so that one or more of thefollowing amino acid substitutions have been made: Arg (A) in position285 has been replaced by Gln (Q); Thr (T) in position 296 has beenreplaced by Ala (A); Leu (L) in position 298 has been replaced by Ala(A); Leu (L) in position 298 has been replaced by Val (V); Ser (S) inposition 314 has been replaced by Asn (N); Asn (N) in position 322 hasbeen replaced by Lys (K); Met (M) in position 327 has been replaced byVal (V); Phe (F) in position 335 has been replaced by Ala (A); Phe (F)in position 335 has been replaced by Leu (L); Tyr (Y) in position 337has been replaced by Ala (A); Lys (K) in position 339 has been replacedby Arg (R); Lys (K) in position 339 has been replaced by Ala (A); Gln(Q) in position 349 has been replaced by Glu (E); Ile (I) in position362 has been replaced by Val (V); Arg (R) in position 365 has beenreplaced by His (H); Phe (F) in position 366 has been replaced by Tyr(Y); and Pro (P) in position 375 has been replaced by Leu (L), with theproviso that the CH3 region has been modified so that it does notcomprise any acceptor sites for N-linked glycosylation.

In a further embodiment hereof, one or more of the of the followingamino acid substitutions have been made: Arg (R) in position 285 hasbeen replaced by Gln (Q); Ser (S) in position 314 has been replaced byAsn (N); Asn (N) in position 322 has been replaced by Lys (K); Met (M)in position 327 has been replaced by Val (V); Lys (K) in position 339has been replaced by Arg (R); Gln (Q) in position 349 has been replacedby Glu (E).; IIe (I) in position 352 has been replaced by Val (V); Arg(R) in position 365 has been replaced by His (H); Phe (F) in position366 has been replaced by Tyr (Y); and Pro (P) in position 375 has beenreplaced by Leu (L). In an even further embodiment:

-   (i) Arg (R) in position 285 has been replaced by Gln (Q),-   (ii) Arg (R) in position 285 has been replaced by Gln (Q); and    Pro (P) in position 375 has been replaced by Leu (L), or-   (iii) all ten substitutions as defined above have been made.

In another further embodiment hereof, the monovalent antibody furthercomprises the CH1 and/or CH2 regions as set forth in SEQ ID NO: 9, withthe proviso that the CH2 region has been modified so that it does notcomprise any acceptor sites for N-linked glycosylation.

In further embodiments, the monovalent antibody according to theinvention has been further modified e.g. in the CH2 and/or CH3 region,for example, to reduce the ability of the monovalent antibody todimerize or to improve the pharmacokinetic profile, e.g. via improvingthe binding to FcRn.

Examples of such modifications include the following substitutions(reference is here made to IgG4 residues given in SEQ ID NO:4, but thesame substitutions may be made in corresponding residues in otherisotypes, such as IgG1. These corresponding residues may be found bysimply alignment of the sequence): in the CH3 region: T234A, L236A,L236V, F273A, F273L, Y275A, E225A, D267A, L236E, L236G, F273D, F273T,Y275E, and in the CH2 region: T118Q, M296L, M120Y, S122T, T124E, N302A,T175A, E248A, N302A. Two or more of the above mentioned substitutionsmade combined to obtain the combined effects.

Thus, in one embodiment, the monovalent antibody comprises the CH3region as set forth in SEQ ID NO: 4.

However, in another embodiment, the monovalent antibody comprises theCH3 region as set forth in SEQ ID NO: 4, but:

-   Glu (E) in position 225 has been replaced by Ala (A), and/or-   Thr (T) in position 234 has been replaced by Ala (A), and/or-   Leu (L) in position 236 has been replaced by Ala (A), Val (V),    Gln (E) or Gly (G), and/or-   Asp (D) in position 267 has been replaced by Ala (A), and/or-   Phe (F) in position 273 has been replaced by Ala (A) or Leu (L).-   Tyr (Y) in position 275 has been replaced by Ala (A).

In another embodiment, the monovalent antibody comprises the CH3 regionas set forth in SEQ ID NO: 4, but:

-   Glu (E) in position 225 has been replaced by Ala (A), and/or-   Thr (T) in position 234 has been replaced by Ala (A), and/or-   Leu (L) in position 236 has been replaced by Ala (A), Val (V),    Glu (E) or Gly (G), and/or-   Asp (D) in position 267 has been replaced by Ala (A), and/or-   Phe (F) in position 273 has been replaced by Asp (D) and Tyr (Y) in    position 275 has been replaced by Glu (E).

In another embodiment, the monovalent antibody comprises the CH3 regionas set forth in SEQ ID NO: 4, but:

-   Glu (E) in position 225 has been replaced by Ala (A), and/or-   Thr (T) in position 234 has been replaced by Ala (A), and/or-   Leu (L) in position 236 has been replaced by Ala (A), Val (V),    Glu (E) or Gly (G), and/or-   Asp (D) in position 267 has been replaced by Ala (A), and/or-   Phe (F) in position 273 has been replaced by Thr (T) and Tyr (Y) in    position 275 has been replaced by Glu (E).

In one embodiment, the monovalent antibody comprises the CH2 region asset forth in SEQ ID NO: 4, but wherein Thr (T) in position 118 has beenreplaced by Gln (Q) and/or Met (M) in position 296 has been replaced byLeu (L).

In another embodiment, the monovalent antibody comprises the CH2 regionas set forth in SEQ ID NO: 4, but wherein one, two or all three of thefollowing substitutions have been made: Met (M) in position 120 has beenreplaced by Tyr (Y); Ser (S) in position 122 has been replaced by Thr(T); and Thr (T) in position 124 has been replaced by Glu (E).

In another embodiment, the monovalent antibody comprises the CH2 regionas set forth in SEQ ID NO: 4, but wherein Asn (N) in position 302 hasbeen replaced by Ala (A).

In a yet other embodiment, the monovalent antibody comprises the CH2region as set forth in SEQ ID NO: 4, but wherein Asn (N) in position 302has been replaced by Ala (A) and Thr (T) in position 175 has beenreplaced by Ala (A) and Glu (E) in position 248 has been replaced by Ala(A).

In an even further different embodiment, the antibody of the inventioncomprises the CH3 region as set forth in SEQ ID NO: 4, and wherein theCH3 region has been modified so that one or more of the following aminoacid substitutions have been made: Thr (T) in position 234 has beenreplaced by Ala (A); Leu (L) in position 236 has been replaced by Ala(A); Len (L) in position 236 has been replaced by Val (V); Phe (F) inposition 273 has been replaced by Ala (A); Phe (F) in position 273 hasbeen replaced by Leu (L); Tyr (Y) in position 275 has been replaced byAla (A); Arg (R) in position 277 has been replaced by Ala (A).

Preferred substitutions include; replacement of Leu (L.) in position 236by Val (V), replacement of Phe (F) in position 273 by Ala (A) andreplacement of of Tyr (Y) in position 275 by Ala (A).

In one embodiment of the invention, the monovalent antibody does notbind to the synthetic antigen (Tyr, Glu)-Ala-Lys.

The hinge region is a region of an antibody situated between the CH1 andCH2 regions of the constant domain of the heavy chain. The extent of thehinge region is determined by the separate exon, which encodes the hingeregion. The hinge region is normally involved in participating inensuring the correct assembly of the four peptide chains of an antibodyinto the traditional tetrameric form via the formation of disulphidebonds, or bridges, between one or more cysteine residues in the hingeregion of one of the heavy chains and one or more cysteine residues inthe hinge region of the other heavy chain. A modification of the hingeregion so that none of the amino acid residues in the hinge region arecapable of participating in the formation of disulphide bonds may thusfor instance comprise the deletion and/or substitution of the cysteineresidues present in the unmodified hinge region. A region correspondingto the hinge region should for the purpose of this specification beconstrued to mean the region between region CH1 and CH2 of a heavy chainof an antibody. In the context of the present invention, such a regionmay also comprise no amino acid residues at all, corresponding to adeletion of the hinge region, resulting in the CH1 and CH2 regions beingconnected to each other without any intervening amino acid residues.Such a region may also comprise only one or a few amino acid residues,which residues need not be the amino acid residues present in the N-orC-terminal of the original hinge region.

Accordingly, in one embodiment of the antibody of the invention, the CHregion has been modified such that the region corresponding to the hingeregion of the CH region does not comprise any cysteine residues. Inanother embodiment, the CH region has been modified such that at leastall cysteine residues have been deleted and/or substituted with otheramino acid residues. In a further embodiment, the CH region has beenmodified such that the cysteine residues of the hinge region have beensubstituted with amino acid residues that have an uncharged polar sidechain or a nonpolar side chain. Preferably, the amino acids withuncharged polar side chains are independently selected from asparagine,glutamine, serine, threonine, tyrosine, and tryptophan, and the aminoacid with the nonpolar side chain are independently selected fromalanine, valine, leucine, isoleucine, proline, phenylalanine, andmethionine.

In an even further embodiment, the monovalent antibody is a human IgG4,wherein the amino acids corresponding to amino acids 106 and 109 of theCH sequence of SEQ ID No: 2 have been deleted.

In a yet further embodiment, the monovalent antibody is a human IgG4,wherein one of the amino acid residues corresponding to amino acidresidues 106 and 109 of the sequence of SEQ ID No: 2 has beensubstituted with an amino acid residue different from cysteine, and theother of the amino acid residues corresponding to amino acid residues106 and 109 of the sequence of SEQ ID No: 2 has been deleted.

In a yet further embodiment, the amino acid residue corresponding toamino acid residue 106 has been substituted with an amino acid residuedifferent from cysteine, and the amino acid residue corresponding toamino acid residue 109 has been deleted.

In a yet further embodiment, the amino acid residue corresponding toamino acid residue 106 has been deleted, and the amino acid residuecorresponding to amino acid residue 109 has been substituted with anamino acid residue different from cysteine.

In a yet further embodiment, the monovalent antibody is a human IgG4,wherein at least the amino acid residues corresponding to amino acidresidues 106 to 109 of the CH sequence of SEQ ID No: 2 have beendeleted.

In a yet further embodiment, the monovalent antibody is a human IgG4,wherein at least the amino acid residues corresponding to amino acidresidues 99 to 110 of the sequence of SEQ ID No: 2 have been deleted.

In a yet further embodiment, the CH region comprises the amino acidsequence of SEQ ID No: 4.

In a yet even further embodiment, the monovalent antibody is a humanIgG4, wherein the CH region has been modified such that the entire hingeregion has been deleted.

In a further embodiment, the sequence of the antibody has been modifiedso that it does not comprise any acceptor sites for N-linkedglycosylation. In a further embodiment hereof, the NST acceptor site forN-linked glycosylation in the CH2 region has been modified to a sequenceselected from the group consisting of: GST, MST, CSE, DSE, DSP, ESP,GSP, NSE, NSE, PSP and SSE.

In one embodiment, the monovalent antibody of the invention ismonovalent in the presence of physiological concentrations of polyclonalhuman IgG.

The antibodies of the present invention has the advantage of having along half-life in vivo, leading to a longer therapeutic window, ascompared to e.g. a FAB fragment of the same antibody which has aconsiderably shorter half-life in vivo.

Further, due to the long half-life and small size, the monovalentantibodies of the invention will have a potential having a betterdistribution in vivo, in example by being able to penetrate solidtumors. This leads to a great use potential of the monovalent antibodiesof the invention, e.g. for treatment of cancer, since the antibodies ofthe invention could be used either to inhibit a target molecule, or as atarget specific delivery mechanism for other drugs that would treat thedisease.

Accordingly, in one embodiment, the monovalent antibody of the inventionhas a plasma concentration above 10 μg/ml for more than 7 days whenadministered in vivo at a dose of 4 mg per kg, as measured in anphermacokinetic study in SCID mice (for instance as shown in theWO2007059782). The clearance rate of a monovalent antibody of theinvention may be measured by use of pharmacokinetic methods as it isknown in the art. The antibody may for instance be injectedintravenously (other routes such as i.p. or i,m, may also be used) in ahuman or animal after which blood samples are drawn by venipuncture atseveral time points, for instance 1 hour, 4 hours, 24 hours, 3 days, 7days, 14 days, 21 days and 28 days after initial injection). Theconcentration of antibody in the serum is determined by an appropriateassay such as ELISA. Pharmacokinetic analysis can performed as known inthe art and described in WO2007059782. Monovalent antibodies of theinvention may have a plasma residence time, which is as much as 100times longer than the plasma residence time of for instance Fabfragments which are frequently used as monovalent antibodies.

In one embodiment, a monovalent antibody of the invention has a plasmaclearance, which is more than 10 times slower than the plasma clearanceof a F(ab′)₂ fragment, which has a comparable molecular size. This maybe an indication of the capability of the antibodies of the invention tobind to FcRn. FcRn is a major histocompatibility complex class l-relatedreceptor and plays a role in the passive delivery of immunoglobulin(1g)Gs from mother to young and in the regulation of serum IgG levels byprotecting IgG from intracellular degradation (Ghetie V et al., Annu Revimmunol. 18, 739-66 (2000)). In one embodiment, the F(ab′)₂ fragment isdirected at the same antigen as the monovalent antibody of theinvention. In one embodiment, the F(ab′)₂ fragment is directed at thesame epitope as the monovalent antibody of the invention, in oneembodiment, the VH region and the VL region of the F(ab′)₂ fragment areidentical to the VH region and the VL region of the monovalent antibodyof the invention.

In one embodiment, a monovalent antibody of the invention has ahalt-life of at least 5 days when administered in vivo. The half-life ofa monovalent antibody of the invention may be measured by any methodknown in the art, for instance as described above.

In one embodiment, a monovalent antibody of the invention has ahalf-life of at least 5 days and up to 14 days, when administered invivo.

In one embodiment, the monovalent antibody of the invention has ahalf-life of at least 5 days and up to 21 days, when administered invivo.

In an even further embodiment, the monovalent antibody has a serumhalf-life of at least 5 days, such as of at least 14 days, for exampleof from 5 and up to 21 days when administered in vivo to a human beingor a SCID mouse.

In one embodiment, the monovalent antibody of the invention binds to atumor antigen with a dissociation constant (k_(d)) of 10⁻⁷ M or less,such as 10⁻⁸ M or less.

In another embodiment, the monovalent antibody of the invention binds toa cell surface receptor with a dissociation constant (k_(d)) of 10⁻⁷ Mor less, such as 10⁻⁸ M or less, which cell surface receptor isactivated upon receptor dirnerization.

In a further embodiment, the monovalent antibody binds to a target witha dissociation constant (k_(d)) of 10⁻⁷ M or less, such as 10⁻⁸ M orless, which target is selected from: erythropoietin, beta-amyloid,thrombopoletin, interferon-alpha (2a and 2b), -beta (1b), -gamma, TNFR I(CD120a), TNFR II (CD120b), IL-1R type 1 (CD121a), IL-1R type 2(CD121b), IL-2, IL.2R (CD25), 1L-2R-beta (CD123), IL-3, IL-4, IL-3R(CD123), IL-4R (CD124), IL-5SR (CD125), IL-6R-alpha (CD126), -beta (CD130), IL-10, IL-11, 1IL-15BP, IL-15R, IL-20, 1IL-21, TCR variablechain, RANK, RANK-L, CTLA4, CXCR4R, CCR5R, TGF-beta1, -beta2, beta3,G-CSF, GM CSF, MIF-R (CD74), M-CSF-R (CD115), GM-CSFB (CD116), solubleFcRI, sFcRII, sFcRIII, FcRn, Factor VII, Factor VIII, Factor IX, VEGF,VEGFxxxb, anti-psychotic drugs, anti-depressant drugs, anti-Parkinsondrugs, anti-seizure agents, neuromuscular blocking drugs, anti-epilepticdrugs, adrenocorticosteroids, insulin, proteins or enzymes involved inregulation of insulin, incretins (GIP and GLP-1) or drugs mimickingincretin action such as Exenaticie and sitagliptin, thyroid hormones,growth hormone, ACTH, oestrogen, testosterone, anti-diuretic hormone,diuretics, blood products such as heparin and FPO, beta-blocking agents,cytotoxic agents, anti-viral drugs, anti-bacterial agents, anti-fungalagents, anti-parasitic drugs, anti-coagulation drugs, anti-inflammatorydrugs, anti-asthma drugs, anti-COPD drugs, Viagra, opiates, morphine,vitamins (such as vitamin C for conservation), hormones involved inpregnancy such as LH and FSH, hormones involved in sex changes,anti-conceptives and antibodies.

In one embodiment, a monovalent antibody of the invention specificallybinds a cell surface receptor that is activated upon receptordimerization. Monovalent antibodies, such as the monovalent antibodiesof the invention, may often be useful in the treatment of diseases ordisorders, where receptor activation is undesirable, since the antibodymolecules of the inventions due to their monovalent nature are unable toinduce such dimerization and thereby such activation. Without beinglimited to specific receptors, examples of such receptors could beerb-B1, erb-B2, erb-B3, erb-B4 and members of the ephrins and ephrinreceptors such as ephrin-A1 through A6, ephA1 through A8, ephrin B1through B3 and eph-B1 through eph-B6.

In one embodiment, a monovalent antibody of the invention, when bound toa target molecule, inhibits target molecule multimerization (such asdimerization). Again, monovalent antibodies, such as the monovalentantibodies of the invention, may often be useful in the treatment ofdiseases or disorders, where multimerization of the target antigen isundesirable, since the antibody molecules of the inventions due to theirmonovalent nature are unable to induce such multimerization. In the caseof soluble antigens, multimerization may form undesirable immunecomplexes. Without being limited to specific targets, examples of suchtargets could be Toll-like receptors such as TLR-3 and TLR-9, orangiopoietin-1, or angiopoietin-2, or TNF receptor family members suchas CD30, CD40 and CD95.

In one embodiment, a monovalent antibody of the invention is aninhibitor of TNF-alpha. In one embodiment of the invention; themonovalent antibody of the invention is a monovalent form of adalimumab,etanercept, or infliximab.

In a further embodiment, the monovalent antibody binds to a target witha dissociation constant (k_(d)) of 10⁻⁷ M or less, such as 10⁻⁸ M. orless, which target is selected from VEGF, c-Met, CD20, CD38, IL 8, CD25,CD74, FcalphaRI, FcepsilonRI, acetyl choline receptor, fas, fasL, TRAIL,hepatitis virus, hepatitis C virus, envelope E2 of hepatitis C virus,tissue factor, a complex of tissue factor and Factor VII, EGFr, CD4, andGD28.

In one embodiment, an anti-VEGF monovalent antibody is used fortreatment of AMD (acute macular degeneration), and other diseases.

In one embodiment, the anti-VEGF monovalent antibody used is amonovalent form of bevacizumab (Avastin).

In an even further embodiment, the monovalent antibody is a human IgG4antibody and which binds to c-Met with a dissociation constant (k_(d))of 10⁻⁷ M or less, such as 10⁻⁸ M or less.′

In one embodiment, a monovalent antibody of the invention is incapableof effector binding. The expression “incapable of effector binding” or“inability of effector binding” in the present context means that amonovalent antibody of the invention is incapable of binding to the C1qcomponent of the first component of complement (C1) and therefore isunable of activating the classical pathway of complement mediatedcytotoxicity. In addition, the monovalent antibodies of the inventionare unable to interact with Fc receptors and may therefore be unable totrigger Fc receptor-mediated effector functions such as phagocytosis,cell activation, induction of cytokine release.

In one embodiment, a monovalent antibody of the invention is produced byuse of recombinant DNA technologies. Antibodies may be produced usingrecombinant eukaryotic host cells, such as Chinese hamster ovary (CHO)cells, NS/0 cells, HEK293 cells, insect cells, plant cells, or fungi,including yeast cells. Both stable as well as transient systems may beused for this purpose. Transfection may be done using plasmid expressionvectors by a number of established methods, such as electroporation,lipofection or nueleofection. Alternatively, infection may be used toexpress proteins encoded by recombinant viruses such as adeno, vacciniaor baculoviruses. Another method may be to use transgenic animals forproduction of antibodies.

Thus, in a further main aspect, the invention relates to a nucleic acidconstruct encoding the monovalent antibody of the invention as describedherein. In one embodiment, said nucleic acid construct is an expressionvector.

Furthermore, the invention relates to a method of preparing a monovalentantibody according to the invention comprising culturing a host cellcomprising a nucleic acid construct according to invention, so that themonovalent antibody is produced, and recovering the said monovalentantibody from the cell culture.

A DNA sequence encoding the antibody may be prepared synthetically byestablished standard methods. The DNA sequence may then be inserted intoa recombinant expression vector, which may be any vector, which mayconveniently be subjected to recombinant DNA procedures. The choice ofvector will often depend on the host cell into which it is to beintroduced. Thus, the vector may be an autonomously replicating vector,i.e. a vector that exists as an extrachromosomal entity, the replicationof which is independent of chromosomal replication, for instance aplasmid. Alternatively, the vector may be one which, when introducedinto a host cell, is integrated into the host cell genome and replicatedtogether with the chromosome(s) into which it has been integrated. Inthe vector, a DNA sequence encoding the antibody should be operablyconnected to a suitable promoter sequence. The coding DNA sequence mayalso be operably connected to a suitable terminator and the vector mayfurther comprise elements such as polyadenylation signals (for instancefrom SV40 or the adenovirus 5 Elb region), transcriptional enhancersequences (for instance the SV40 enhancer) and translational enhancersequences (for instance the ones encoding adenovirus VA RNAs). Othersuch signals and enhancers are known in the art.

To obtain recombinant monovalent antibodies of the invention, the DNAsequences encoding different parts of the polypeptide chain(s) of theantibody may be individually expressed in a host cell, or may be fused,giving a DNA construct encoding the fusion polypeptide, such as apolypeptide comprising both light and heavy chains, inserted into arecombinant expression vector, and expressed in host cells.

Thus, in a further aspect, the invention relates to a host cellcomprising a nucleic acid according to the invention.

The invention also relate to a non-human transgenic animal comprising anucleic acid construct according to the invention.

The host cell into which the expression vector may be introduced, may beany cell which is capable of expression of full-length proteins, and mayfor instance be a prokaryotic or eukaryotic cell, such as yeast, insector mammalian cells. Examples of suitable mammalian cell lines are theHEK293 (ATCC CRL-1573), COS (ATCC CRL-1650), BHK (ATCC CRL-1632, ATCCCCL-10), NS/0 (ECACC 85110503) or CHO (ATCC CCL-61) cell lines. Othersuitable cell lines are known in the art. In one embodiment, theexpression system is a mammalian expression system, such as a mammaliancell expression system comprising various clonal variations of HEK293cells.

Methods of transfecting mammalian cells and expressing DNA sequencesintroduced in the cells are well known in the art. To obtain amonovalent antibody of the invention, host cells of the expressionsystem may in one embodiment to be cotransfected with two expressionvectors simultaneously, wherein first of said two expression vectorscomprises a DNA sequence encoding the heavy chain of the antibody, andsecond of said two expression vectors comprises a DNA sequence encodingthe light chain of the antibody. The two sequences may also be presenton the same expression vector, or they may be fused giving a DNAconstruct encoding the fusion polypeptide, such as a polypeptidecomprising both light and heavy chains.

The recombinantly produced monovalent antibody may then be recoveredfrom the culture medium by conventional procedures including separatingthe host cells from the medium by centrifugation or filtration,precipitating the proteinaceous components of the supernatant orfiltrate by means of a salt, for instance ammonium sulphate,purification by a variety of chromatographic procedures, for instanceHPLC, ion exchange chromatography, affinity chromatography, Protein Achromatography, Protein G chromatography, or the like.

The present invention also relates to a method of preparing a monovalentantibody of the invention, wherein said method comprises the steps of:

-   -   (a) culturing a host cell comprising a nucleic acid encoding        said monovalent antibody; and    -   (b) recovering the monovalent antibody from the host cell        culture.

In one embodiment, said host cell is a prokaryotic host cell. In oneembodiment, the host cell is an E. coli cell. In one embodiment, the E.coli cells are of a strain deficient in endogenous protease activities.

In one embodiment, said host cell is a eukaryotic cell. In oneembodiment, the host cell is a HEK-293F cell. In another embodiment, thehost cell is a CHO cell.

In one embodiment, the monovalent antibody is recovered from culturemedium. In another embodiment, the monovalent antibody is recovered fromcell lysate:

In a further main aspect, the invention relates to a pharmaceuticalcomposition comprising the monovalent antibody according to theinvention. In one embodiment, the composition further comprises one ormore further therapeutic agents described herein.

The pharmaceutical compositions may be formulated with pharmaceuticallyacceptable carriers or diluents as well as any other known adjuvants andexcipients in accordance with conventional techniques such as thosedisclosed in Remington: The Science and Practice of Pharmacy, 19^(th)Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995. As usedherein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonicity agents, antioxidants and absorption delaying agents,and the like that are physiologically compatible.

The pharmaceutical composition may be administered by any suitable routeand mode. As will be appreciated by the skilled artisan, the routeand/or mode of administration will vary depending upon the desiredresults.

In one embodiment, the pharmaceutical composition is suitable forparenteral administration. The phrase “parenteral administration” meansmodes of administration other than enteral and topical administration,usually by injection, and includes, without limitation, intravenous,intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,epidural and intrasternal injection and infusion. In one embodiment thepharmaceutical composition is administered by intravenous orsubcutaneous injection or infusion.

Regardless of the route of administration selected, the monovalentantibodies of the present invention, which may be′ used in the form of apharmaceutically acceptable salt or in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Dosage regimens are adjusted to provide the optimum desired response(for instance a therapeutic response). For example, a single bolus maybe administered, several divided doses may be administered over time orthe dose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofmonovalent antibody calculated to produce the desired therapeutic effectin association with the required pharmaceutical carrier. Thespecification for the dosage unit forms of the invention are dictated byand directly dependent on (a) the unique characteristics of themonovalent antibody and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch a monovalent antibody for the treatment of sensitivity inindividuals.

Actual dosage levels of the monovalent antibodies in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration. The selected dosage level will depend upon avariety of pharmacokinetic factors including the activity of theparticular monovalent antibodies of the present invention employed, theroute of administration, the time of administration, the rate ofexcretion of the particular monovalent antibody being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved. In general, a suitable doseof a pharmaceutical composition of the invention will be that amount ofthe monovalent antibody which is the lowest dose effective to produce atherapeutic effect. Such an effective dose will generally depend uponthe factors described above. As another example, the physician orveterinarian may start with a high loading dose followed by repeatedadministration of lower doses to rapidly build up a therapeuticallyeffective dose and maintain it over longer periods of time.

A pharmaceutical composition of the invention may contain one or acombination of different monovalent antibodies of the invention. Thus,in a further embodiment, the pharmaceutical compositions include acombination of multiple (for instance two or more) monovalent antibodiesof the invention which act by different mechanisms. The monovalentantibodies may also be thus combined with divalent antibodies.

The monovalent antibody of the present invention have numerous in vitroand in vivo diagnostic and therapeutic utilities involving the diagnosisand treatment of disorders involving cells expressing the antigen whichthe antibody can recognize and bind to. In certain pathologicalconditions, it is necessary and/or desirable to utilize monovalentantibodies. Also, in some instances, it is preferred that a therapeuticantibody effects its therapeutic action without involving immunesystem-mediated activities, such as the effector functions, ADCC,phagocytosis and CDC. In such situations, it is desirable to generateforms of antibodies in which such activities are substantially reducedor eliminated. It is also advantageous if the antibody is of a form thatcan be made efficiently and with high yield. The present inventionprovides such antibodies, which may be used for a variety of purposes,for example as therapeutics, prophylactics and diagnostics.

In one embodiment, a monovalent antibody of the invention is directed toCD74 and inhibits MlF-induced signaling, but lacks Fc-mediated effectorfunctions.

In one embodiment, a monovalent antibody of the invention may preventbinding of a virus or other pathogen to its receptor, such as inhibitionof HIV binding to CD4 or coreceptor such as CCR5 or CXCR4.

The scientific literature is abundant with examples of targets, wherethe binding of antibodies against said target, or specific epitopes ofsaid target, is shown to have, or is expected to have, a therapeuticeffect. Given the teaching of this specification and as describedelsewhere herein, it is within the skill of a person skilled in the artto determine, whether the use of a monovalent antibody, such as amonovalent antibody of the present invention, against such targets wouldbe expected to produce the therapeutic effect.

Accordingly, in a further aspect, the invention relates to themonovalent antibody according to the invention as described herein foruse as a medicament.

In another aspect, the invention relates to the monovalent antibodyaccording to the invention for use in the treatment of cancer.

In another aspect, the invention relates to the monovalent antibodyaccording to the invention for use in the treatment of an inflammatorycondition.

In another aspect, the invention relates to the monovalent antibodyaccording to the invention for use in the treatment of an auto(immune)disorder.

In another aspect, the invention relates to the monovalent antibodyaccording to the invention for use in the treatment of a disorderinvolving undesired angiogenesis.

In a further aspect, the invention relates to the monovalent antibodyaccording to the invention for use in the treatment of a disease ordisorder, which disease or disorder is treatable by administration of anantibody against a certain target, wherein the involvement of immunesystem-mediated activities is not necessary or is undesirable forachieving the effects of the administration of the antibody, and whereinsaid antibody specifically binds said antigen.

In a further aspect, the invention relates to the monovalent antibodyaccording to the invention for use in the treatment of a disease ordisorder, which disease or disorder is treatable by blocking orinhibiting a soluble antigen, wherein multimerization of said antigenmay form undesirable immune complexes, and wherein said antibodyspecifically binds said antigen.

In a further aspect, the invention relates to the monovalent antibodyaccording to the invention for use in the treatment of a disease ordisorder, which disease or disorder is treatable by blocking orinhibiting a cell membrane bound receptor, wherein said receptor may beactivated by dimerization of said receptor, and wherein said antibodyspecifically binds said receptor.

In one embodiment of any of the above treatments, the treatmentcomprises administering one or more further therapeutic agents.

Similarly, the invention relates to the use of the monovalent antibodyaccording to the invention as described herein as a medicament.

The invention also relates to a method of treating a disease or disorderas defined herein, wherein said method comprises administering to asubject in need of such treatment a therapeutically effective amount ofa monovalent antibody according the invention, a pharmaceuticalcomposition according to the invention or a nucleic acid constructaccording to the invention. In one embodiment, the treatment comprisesadministering one or more further therapeutic agents.

Furthermore, the invention relates to the use of the monovalent antibodyaccording to the invention in the preparation of a medicament for thetreatment of a disease or disorder as defined herein.

In one embodiment of the invention, the disease or disorder to betreated is treatable by interference with cell activation through FcαRI,by interference with FcαRI function, by inhibition of subsequent FcαRIactivated IgE mediated responses, or by binding of soluble FcαRI. In oneembodiment of the invention, the monovalent antibody is directed againstFcαRI and induces apoptosis of FcαRI expressing cells. In oneembodiment, such disease or disorder may for instance be allergic asthmaor other allergic diseases such as allergic rhinitis, seasonal/perennialallergies, hay fever, nasal allergies, atopic dermatitis, eczema, hives,urticaria, contact allergies, allergic conjunctivitis, ocular allergies,food and drug allergies, latex allergies, or insect allergies, or IgAnephropathy, such as IgA pemphigus. In one such embodiment, themonovalent antibody of the invention is directed at FcαRI. Suchmonovalent antibodies may also be used for in vitro or in vivo screeningfor FcαRI in sample or patient or in an immunotoxin or radiolabelapproach to treating these diseases and disorders.

In one embodiment of the invention, the disease or disorder to betreated is treatable by downregulating Fc receptor γ-chain mediatedsignaling through FcεR1 or Fcy receptors. Monomeric binding of antibodyto FcαRI is known to effect such inhibition. Monovalent antibodies maythus be used to inhibit immune activation through a range of Fcreceptors including Fcγ, Fcα and Fcε receptors. Thus, in one embodiment,the monovalent antibody of the invention may bind an Fcα, Fcα, or Fcγreceptor, such as CD32b.

In one such embodiment, the monovalent antibody of the invention isdirected at CD25. Such monovalent antibodies may also be used for invitro or in vivo screening for CD25 in sample or patient or in animmunotoxin or radiolabel approach to treating these diseases anddisorders.

In one embodiment of the invention, the disease or disorder to betreated is treatable by antagonizing and/or inhibiting IL-15 or IL15receptor functions. In one embodiment, such disease or disorder may forinstance be arthritides, gout, connective, neurological,gastrointestinal, hepatic, allergic, hematologic, skin, pulmonary,malignant, endocrinological, vascular, infectious, kidney, cardiac,circulatory, metabolic, bone, and muscle disorders. In one suchembodiment, the monovalent antibody of the invention is directed atIL-15. Such monovalent antibodies may also be used for in vitro or invivo screening for IL-15 in a sample or patient or in an immunotoxin orradiolabel approach to treating these diseases and disorders.

In one embodiment of the invention, the disease or disorder to betreated is treatable by interfering with CD20 activity, by depleting Bcells, interfering with B cell growth and/or proliferation through forinstance an immunotoxin or radiolabel approach. In one embodiment, suchdisease or disorder may for instance be rheumatoid arthritis,(auto)immune and inflammatory disorders (as described above for IL-8related diseases and disorders), non-Hodgkin's lymphoma, B-CLL, lymphoidneoplasms, malignancies and hematological disorders, infectious diseasesand connective, neurological, gastrointestinal, hepatic, allergic,hematologic, skin, pulmonary, malignant, endocrinological, vascular,infectious, kidney, cardiac, circulatory, metabolic, bone and muscledisorders, and immune mediated cytopenia.

In one such embodiment, the monovalent antibody of the invention isdirected at CD20. Such monovalent antibodies may also be used for invitro or in vivo screening for CD20 in a sample or patient.

In one embodiment of the invention, the disease or disorder to betreated is treatable by interfering with CD38 activity, by depletingCD38 expressing cells, interfering with C03⁺cell growth and/orproliferation through for instance an immunotoxin or radiolabelapproach.

In one embodiment of the invention, the disease or disorder to betreated is treatable by blocking ligand-EGFr interaction, blocking EGFrfunction, depletion of EGFr expressing cells/interference with EGFr+cell growth and/or proliferation through for instance an immunotoxin orradiolabel approach.

In one such embodiment, the monovalent antibody of the invention isdirected at EGFr. Such monovalent antibodies may also be used for invitro or in vivo screening for EGFr in a sample or patient.

In one embodiment of the invention, the disease or disorder to betreated is treatable by interfering with CD4 function, depletion of CD4expressing cells/interference with CD4+ cell growth and/or proliferationthrough for instance an immunotoxin or radiolabel approach. In oneembodiment, such disease or disorder may for instance be rheumatoidarthritis, (auto)immune and inflammatory disorders (as described abovefor IL-5 related diseases and disorders), cutaneous T cell lymphomas,non-cutaneous T cell lymphomas, lymphoid neoplasms, malignancies andhematological disorders, infectious diseases, and connective,neurological, gastrointestinal, hepatic, allergic, hematologic, skin,pulmonary, malignant, endocrinological, vascular, infectious, kidney,cardiac, circulatory, metabolic, bone, and muscle disorders, and immunemediated cytopenia.

In one such embodiment, the monovalent antibody of the invention isdirected at CD4. Such monovalent antibodies may also be used for invitro or in vivo screening for CD4 in a sample or patient.

In one embodiment of the invention, a monovalent antibody directed atCD4 is used for treatment of HIV infection, or for the treatment ofAIDS.

In one embodiment of the invention, the monovalent antibodies of theinvention are monovalent antibodies of the CD4 antibodies disclosed inWO97/13852.

In one embodiment of the invention, the disease or disorder to betreated is treatable by antagonizing and/or inhibiting CD28 functions,such as preventing of co-stimulatory signals needed in T cellactivation. In one embodiment, such disease or disorder may for instancebe an inflammatory, autoimmune and immune disorder as indicated above.In one such embodiment, the monovalent antibody of the invention isdirected at CD28.

In one embodiment of the invention, the disease or disorder to betreated is treatable by altering Tissue Factor functions, such asaltering coagulation or inhibition of tissue factor signalling. In oneembodiment, such disease or disorder may for instance be vasculardiseases, such as myocardial vascular disease, cerebral vasculardisease, retinopathia and macular degeneration, and inflammatorydisorders as indicated above.

In one embodiment of the invention, the monovalent antibodies aredirected at Tissue factor, or at a complex of Factor VII and TissueFactor.

In one embodiment of the invention, the disease or disorder to betreated is treatable by interfering with Hepatitis C Virus (HCV)infection. In one such embodiment, the monovalent antibody of theinvention is directed at HCV or an HCV receptor such as CD81.

In one embodiment of the invention, the monovalent antibody is amonovalent antibody according to the invention of an antibody asdisclosed in WO2000/05266.

In one embodiment of the invention, the disease or disorder to betreated is treatable by prevention of binding of allergen toIgE-sensitized on mast cell. In one embodiment, such disease or disordermay for instance be allergen-immunotherapy of allergic diseases such asasthma, allergic rhinitis, seasonal/perennial allergies, hay fever,nasal allergies, atopic dermatitis, eczema, hives, urticaria, contactallergies, allergic conjunctivitis, ocular allergies, food and drugallergies, latex allergies, and insect allergies.

In one such embodiment, the monovalent antibody(s) of the invention areIgG4 hingeless antibodies directed towards allergen(s).

In certain embodiments, an immunoconjugate comprising a monovalentantibody conjugated with a cytotoxic agent is administered to thepatient. In some embodiments, the immunoconjugate and/or antigen towhich it is bound is/are internalized by the cell, resulting inincreased therapeutic efficacy cf the immunoconjugate in killing thetarget cell to which it binds. In one embodiment, the cytotoxic agenttargets or interferes with nucleic acid in the target cell.

Examples of such cytotoxic agents include any of the chemotherapeuticagents noted herein (such as a maytansinoid or a calicheamicin), aradioactive isotope, or a ribonuclease or a DNA endonuclease.

Monovalent antibodies of the invention may be used either alone or incombination with other compositions in a therapy. For instance, amonovalent antibody of the invention may be co-administered with one ormore other antibodies, such as monovalent antibodies of the presentinvention, one or more chemotherapeutic agent(s) (including cocktails ofchemotherapeutic agents), one or more other cytotoxic agent(s), one ormore anti-angiogenic agent(s), one or more cytokines, one or more growthinhibitory agent(s), one or more anti-inflammatory agent(s), one or moredisease modifying antirheumatic drug(s) (DMARD), or one or moreimmunosuppressive agent(s), depending on the disease or condition to betreated. Where a monovalent antibody of the invention inhibits tumorgrowth, it may be particularly desirable to combine it with one or moreother therapeutic agent(s) which also inhibits tumor growth. Forinstance, anti-VEGF antibodies blocking VEGF activities may be combinedwith anti-ErbB antibodies (for instance Trastuzumab (Herceptin), ananti-HER2 antibody) in a treatment of metastatic breast cancer.Alternatively, or additionally, the patient may receive combinedradiation therapy (for instance external beam irradiation or therapywith a radioactive labeled agent, such as an antibody). Such combinedtherapies noted above include combined administration (where the two ormore agents are included in the same or separate formulations), andseparate administration, in which case, administration of the antibodyof the invention may occur prior to, and/or following, administration ofthe adjunct therapy or therapies.

In one embodiment, the monovalent antibody of the invention is amonovalent form of trastuzumab, for treatment of Her2 positive cancer.

For the prevention or treatment of disease, the appropriate dosage of amonovalent antibody of the invention (when used alone or in combinationwith other agents such as chemotherapeutic agents) will depend on thetype of disease to be treated, the type of antibody, the severity andcourse of the disease, whether the monovalent antibody is administeredfor preventive, therapeutic or diagnostic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The monovalent antibody may besuitably administered to the patient at one time or over a series oftreatments.

Such dosages may be administered intermittently, for instance every weekor every three weeks (for instance such that the patient receives fromabout two to about twenty, for instance about six doses of themonovalent antibody). An initial higher loading dose, followed by one ormore lower doses may be administered. An exemplary dosing regimencomprises administering an initial loading dose of about 4 mg/kg,followed by a weekly maintenance dose of about 2 mg/kg of the monovalentantibody. However, other dosage regimens may be useful. In oneembodiment, the monovalent antibodies of the invention are administeredin a weekly dosage of from 50 mg to 4000 mg, for instance of from 250 mgto 2000 mg, such as for example 300 mg, 500 mg, 700 mg, 1000 mg, 1500 mgor 2000 mg, for up to 8 times, such as from 4 to 6 times. The weeklydosage may be divided into two or three subdosages and administered overmore than one day. For example, a dosage of 300 mg may be administeredover 2 days with 100 mg on day one (1), and 200 mg on day two (2). Adosage of 500 mg may be administered over 3 days with 100 mg on day one(1), 200 mg on day two (2), and 200 mg on day three (3), and a dosage of700 mg may be administered over 3 days with 100 mg on day 1 (one), 300mg on day 2 (two), and 300 mg on day 3 (three).The regimen may berepeated one or more times as necessary, for example, after 6 months or12 months.

The dosage may be determined or adjusted by measuring the amount ofcirculating monovalent antibodies of the invention upon administrationin a biological sample for instance by using anti-idiotypic antibodieswhich target said monovalent antibodies.

In one embodiment, the monovalent antibodies of the invention may beadministered by maintenance therapy, such as, for instance once a weekfor a period of 6 months or more.

In one embodiment, the monovalent antibodies of the invention may beadministered by a regimen including one infusion of a monovalentantibody of the invention followed by an infusion of same monovalentantibody conjugated to a radioisotope. The regimen may be repeated, forinstance 7 to 9 days later.

In another main aspect, the invention relates to the use of a monovalentantibody according to the invention as a diagnostic agent.

As described above, in a further aspect, the invention relates to astabilized IgG4 antibody for use as a medicament, comprising a heavychain and a light chain, wherein said heavy chain comprises a human IgG4constant region having the sequence set forth in SEQ ID NO:2, whereinLys (K) in position 250 has been replaced by Gln (Q) or Glu (E) andwherein the antibody optionally comprises one or more furthersubstitutions, deletions and/or insertions in the constant region as setforth in SEQ ID NO2.

In one embodiment thereof, the human IgG4 constant region has thesequence set forth in SEQ ID NO:2, wherein X1 at position 189 is Leu andX2 at position 289 is Arg. In another embodiment thereof, the human IgG4constant region has the sequence set forth in SEQ ID NO:2, wherein X1 atposition 189 is Leu and X2 at position 289 is Lys. In yet anotherembodiment thereof, the human IgG4 constant region has the sequence setforth in SEQ ID NO:2, wherein X1 at position 189 is Val and X2 atposition 289 is Arg.

In one further aspect, the invention relates to an isolated stabilizedIgG4 antibody for use as a medicament., comprising a heavy chain and alight chain, wherein said heavy chain comprises a human IgG4 constantregion having the sequence set forth in SEQ ID NO:2, wherein Lys (K) inposition 250 has been replaced by Gln (Q) Glu (E) and wherein theantibody optionally comprises one or more further substitutions,deletions and/or insertions in the constant region as set forth in SEQID NO:2.

In one embodiment thereof, the human IgG4 constant region has thesequence set forth in SEQ ID NO:2, wherein X1 at position 189 is Leu andX2 at position :289 is Arg. In another embodiment thereof, the humanIgG4 constant region has the sequence set forth in SEQ ID NO:2, whereinX1 at position 189 is Leu and X2 at position 289 is Lys. In yet anotherembodiment thereof, the human IgG4 constant region has the sequence setforth in SEQ ID NO:2, wherein X1 at position 189 is Val and X2 atposition 289 is Arg.

The stabilized IgG4 antibodies according to the invention have theadvantage that they contain a minimal number of sequence changes in theconstant region as compared to naturally occurring IgG4. This reducesthe risk of immunogenicity when the antibody is used for human therapy.

In one embodiment thereof the stabilized IgG4 antibody does not comprisea Cys-Pro-Pro-Cys sequence in the hinge region.

In one embodiment thereof the CH3 region of the stabilized IgG4 antibodyhas been replaced by the CH3 region of human IgG1, of human IgG2 or ofhuman IgG3.

In one embodiment thereof the stabilized IgG4 antibody Does not comprisea substitution of the Leu (L) residue at the position corresponding to115 by a Glu (E).

In one embodiment thereof the stabilized IgG4 antibody does comprise asubstitution of the Leu (L) residue at the position corresponding to 115by a Glu (E).

In one embodiment thereof the stabilized IgG4 antibody comprises one ormore of the following substitutions an Ala (A) at position 114, an Ala(A) at position 116, an Ala (A) at position 117, an Ala (A) at position177, an Ala (A) or Val (V) at position 198, an Ala (A) at position 200,an Ala (A) or Gln (Q) at position 202.

In one embodiment thereof the stabilized IgG4 antibody comprises a CXPCor CPXC sequence in the hinge region, wherein X can be any amino acidexcept for Pro (P).

In one embodiment thereof the stabilized IgG4 antibody does not comprisean extended IgG3-like, hinge region, such as the extended hinge regionas set forth in FIG. 14.

In one embodiment thereof the stabilized IgG4 antibody comprises a CPSCsequence in the hinge region.

In one embodiment thereof the stabilized IgG4 antibody has less than 25,such as less than 10, e.g. less than 9, 8, 7, 6, 5, 4, 3, or 2substitutions, deletions and/or insertions in the constant region as setforth in SEQ ID NO:2.

Typically, the stabilized IgG4 antibody of the invention has a lowerability to activate effector functions as compared to IgG1 and IgG3. Inone embodiment thereof the antibody is less efficient in mediating CDCand/or ADCC than a corresponding IgG1 or IgG3 antibody having the samevariable regions. Assays for measuring CDC or ADCC activity are wellknown in the art.

In one embodiment thereof the stabilized IgG4 antibody is selected fromthe group consisting of a human monoclonal antibody, a humanizedmonoclonal antibody and a chimeric monoclonal antibody.

In one embodiment thereof the stabilized IgG4 antibody comprises a humankappa light chain.

In one embodiment thereof the stabilized IgG4 antibody comprises a humanlambda light chain.

In one embodiment thereof the stabilized IgG4 antibody is a bivalentantibody, for example an antibody which is bivalent even in the presenceof excess of irrelevant antibodies, as explained in the Examples herein.

In one embodiment thereof the stabilized IgG4 antibody is a full-lengthantibody.

Methods for the production of stabilized IgG4 antibodies are well-knownin the art. In a preferred embodiment, antibodies of the invention aremonoclonal antibodies. Monoclonal antibodies may e.g. be produced by thehybridoma method first described by Kohler et al., Nature 256, 495(1975), or may be produced by recombinant DNA methods. Monoclonalantibodies may also be isolated from phage antibody libraries using thetechniques described in, for example, Clarkson et al., Nature 352,624-628 (1991) and Marks et al., J. Mol. Biol. 222, 581-597 (1991)Monoclonal antibodies may be obtained from any suitable source. Thus,for example, monoclonal antibodies may be obtained from hybridomasprepared from murine splenic B cells obtained from mice immunized withan antigen of interest, for instance in form of cells expressing theantigen on the surface, or a nucleic acid encoding an antigen ofinterest. Monoclonal antibodies may also be obtained from hybridomasderived from antibody-expressing cells of immunized humans or non-humanmammals such as rats, dogs, primates, etc.

Further modifications, such as amino acid substitutions, deletions orinsertion as described above, may be performed using standardrecombinant DNA techniques well-known in the art.

In one embodiment, the stabilized IgG4 antibody of the invention is ahuman antibody.

In a further main aspect, the invention relates to a method forproducing a stabilized IgG4 antibody of the invention, said methodcomprising expressing a nucleic acid construct encoding said antibody ina host cell and optionally purifying said antibody.

In one embodiment, the stabilized IgG4 antibody of the invention islinked to a compound selected from the group consisting of a cytotoxicagent; a radioisotope; a prodrug or drug, such as a taxane; a cytokine;and a chemokine. Methods for linking (conjugating) such compounds to anantibody are well-known in the art. References to suitable methods havebeen given in WO 20041056847 (Genmab).

In one embodiment thereof the stabilized IgG4 antibody is linked to acompound selected from the group consisting of a cytotoxic agent; aradioisotope; a prodrug or drug, such as a taxane; a cytokine; and achemokine.

In a further main aspect, the invention relates to a pharmaceuticalcomposition comprising a stabilized IgG4 antibody as defined hereinabove. The pharmaceutical compositions may be formulated withpharmaceutically acceptable carriers or diluents as well as any otherknown adjuvants and excipients in accordance with conventionaltechniques, such as those disclosed in Remington: The Science andPractice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co.,Easton, Pa., 1995.

In one embodiment, a pharmaceutical composition of the present inventionis administered parenterally. The phrases “parenteral administration”and “administered parenterally” as used herein means modes ofadministration other than enteral and topical administration, usually byinjection, and include epidermal, intravenous, intramuscular,intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, intratendinous, transtracheal,subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid,intraspinal, intracranial, intrathoracic, epidural and intrasternalinjection and infusion.

The stabilized IgG4 antibodies of the invention can be used in thetreatment and/or prevention of a number of diseases, and be directed toan antigen selected from a broad variety of suitable target molecules.

In one embodiment thereof the stabilized IgG4 antibody according to anyone of the above embodiments binds to an antigen selected from the groupconsisting of erythropoietin, beta-amyloid, thrombopoletin,interferon-alpha (2a and 2b), interferon-beta (1b), interferon-gamma,TNFR I (CD120a), TNFR II (CD120b), IL-1R type 1 (CD121a), IL-1R type 2(CD121b), IL-2, IL 2R (CD25), IL2R-beta (CD123), IL-3, IL-4, IL-3R(CD123), IL-4R (CD124), IL-5R (CD125), IL-6R-alpha (CD126), -beta(CD130), IL-8, IL-10, IL-11, IL-15, IL-15BP, IL-15R, IL-20, IL-21, TCRvariable chain, RANK, RANK-L, CTLA4, CXCR4R, CCR5R, TGF-beta 1, -beta2,-beta3, G-CSF, GM-CSF, MIF-R (CD74), M-CSF-R (CD115), GM-CSFB (CD116),soluble FcRI, sFcRII, sFcRIII, FcRn, Factor VII, Factor VIII, Factor IX,VEGF VEGFxxxb, alpha-4 integrin, Cd11a, CD18, CD20, CD38, CD25, CD74,FcalphaRI, FcepsilonRI, acetyl choline receptor, faa, fasL, TRAIL,hepatitis virus, hepatitis C virus, envelope E2 of hepatitis C virus,tissue factor, a complex of tissue factor and Factor VII, EGFr, CD4,CD28, VLA-1, 2, 3, or 4, LFA-1, MAC-1, 1-selectin, PSGL-1, ICAM-1,P-selectin, periostin, CD33 (Siglec 3), Siglec 8, TNF, CCL1, CCL2, CCL3,CCL4, CRL5, CCL11, CCL13, CCL17, CCL18, CCL20, CCL22, CCL26, CCL27,CX3CL1, LIGHT, EGF, VEGF, TGFalpha, HGF, PDGF, NGF, complement or arelated components such as: C1q, C4, C2, C3, C5, C6, C7, C9, MBL, factorB, a Matrix Metallo Protease such as any of MMP1 to MMP28, CD32b, CD200,CD200R, Killer Immunoglobulin-Like Receptors (KIRs), NKG2D and relatedmolecules, leukocyte-associated immunoglobulin-like receptors (LAIRS),1γ49, PD-L2, CD26, BST-2, ML-IAP (melanoma inhibitor of apoptosisprotein), cathepsin D, CD40, CD40R, CD86, a B cell receptor, CD79 , PD-1and a cell receptor.

In one embodiment thereof.

-   (i) the antibody binds to an alpha-4 integrin and is for use in the    treatment of inflammatory and autoimmune diseases, such as    rheumatoid arthritis, multiple sclerosis, inflammatory bowel    disease, asthma and sepsis;-   (ii) the antibody binds to VLA-1, 2, 3, or 4 and is for use in the    treatment of inflammatory and autoimmune diseases, such as    rheumatoid arthritis, multiple sclerosis, inflammatory bowel    disease, asthma, type-1 diabetes, SLE, psoriasis, atopic dermatitis,    COPD and sepsis,-   (iii) the antibody binds to a molecule selected from the group    consisting of LFA-1, MAC-1, l-selectin and PSGL-1 and is for use in    the treatment of inflammatory and autoimmune diseases, such as    rheumatoid arthritis, multiple sclerosis, inflammatory bowel    disease, asthma, type-1 diabetes, SLE, psoriasis, atopic dermatitis,    and COPD;-   (iv) the antibody binds to a molecule selected from the group    consisting of LFA-1, MAC-1, 1-selectin and PSGL-1 and is for use in    the treatment of a disease selected from the group consisting of    ischernia-reperfusion injury, cystic fibrosis, osteomyelitis,    glomerulonepritis, gout and sepsis;-   (v) the antibody binds to CD18 and is for use in the treatment of    inflammatory and autoimmune diseases, such as rheumatoid arthritis,    multiple sclerosis, inflammatory bowel disease, asthma, type-1    diabetes, SLE, psoriasis, atopic dermatitis and COPD;-   (vi) the antibody binds to Cd11a and is for use in the treatment of    inflammatory and autoimmune diseases, such as rheumatoid arthritis,    multiple sclerosis, inflammatory bowel disease, asthma, type-1    diabetes, SLE, psoriasis, atopic dermatitis and COPD;-   (vii) the antibody binds lCAM-1 and is for use in the treatment of    inflammatory and autoimmune diseases, such as rheumatoid arthritis,    multiple sclerosis, inflammatory bowel disease, asthma, type-1    diabetes, SLE, psoriasis, atopic dermatitis and COPD;-   (viii) the antibody binds to P-selectin and is for use in the    treatment of cardiovascular diseases, post-thrombotic vein wall    fibrosis, ischemia reperfusion injury, inflammatory diseases or    sepsis.-   (ix) the antibody binds to periostin and is for use in the treatment    of malignant diseases and/or metastasizing diseases, such as ovary    cancer, endometrial cancer, NSCLC, glioblastoma, brain-related    tumors, breast cancer, OSCC, colon cancer, pancreatic cancer, HNSCC,    kidney cancer, thymoma, lung cancer, skin cancer, larynx cancer,    liver cancer, parotid tumors, gastric cancer, esophagus cancer,    prostate cancer, bladder cancer and cancer of the testis;-   (x) the antibody binds to CD33 (Siglec 3), is optionally coupled to    a toxin, cytotoxic or cytostatic drug, and is for use in the    treatment of tumors expressing CD33 or acute myeloid leukemia:-   (xi) the antibody binds to Siglec 8 and is for use in the treatment    of asthma, inflammatory or autoimmune diseases, such as rheumatoid    arthritis, multiple sclerosis, inflammatory bowel disease, asthma,    type-1 diabetes, SLE, psoriasis, atopic dermatitis and COPD:-   (xii the antibody binds to TNF and is for use in the treatment of    inflammatory and autoimmune diseases, such as rheumatoid arthritis,    multiple sclerosis, inflammatory bowel disease, asthma, type-1    diabetes, SLE, psoriasis, atopic dermatitis, COPD and sepsis;-   (xiii) the antibody binds to CCL1, CCL2, CCL3, CCL4, CCL5, CCL11,    CCL13, CCL17, CCL18, CCL20, CCL22, CCL26, CCL27 or CX3CL1 and is for    use in the treatment of atopic dermatitis, inflammatory and    autoimmune diseases, such as rheumatoid arthritis, multiple    sclerosis, inflammatory bowel disease, asthma, type-1 diabetes, SLE,    psoriasis, COPD and sepsis;-   (xiv) the antibody binds to LIGHT and is for use in the treatment of    a disease selected from the group consisting of: hepatitis,    inflammatory bowel disease, GVHD and inflammation;-   (xv) the antibody binds to EGF, VEGF, TGFalpha HGF and is for use in    the treatment of; malignant diseases, such as solid cancers;-   (xvi) the antibody binds to PDGF and is for use in the treatment of    diseases in which abnormal cell proliferation cell migration and/or    angiogenesis occurs, such as atherosclerosis, fibrosis, and    malignant diseases;-   (xvii) the antibody binds to NGF and is for use in the treatment of    neurological diseases, neurodegenerative diseases, such as    Alzheimer's disease and Parkinson's disease, or cancer, such as    prostate cancer;-   (xviii) the antibody binds to complement or a related components    such as C1q, C4, C2, C3, C5, C6, C8, C9, MBL, or factor B and is for    use in diseases in which complement and related components play a    detrimental role, such as organ transplant rejection, multiple    sclerosis, Gulllain-Barre, syndrome, hemolytic anemia, Paroxysmal    Nocturnal Herrioglobinuria, stroke, heart attacks, burn injuries,    age-related macular degeneration, asthma, lupus, arthritis,    myasthenia gravis, anti-phospholipid syndrome, sepsis and ischemia    reperfusion injury;-   (xix) the antibody binds to a Matrix Metalb Protease such as any of    MMP1 to MMP28 and is for use in the treatment of inflammatory and    autoimmune diseases, cancer, including metastatic cancer; arthritis,    inflammation, cardiovascular diseases, cerebrovascular diseases such    as stroke or cerebral aneurysms, pulmonary diseases such as asthma,    ocular diseases such as corneal wound healing or degenerative    genetic eye diseases, gastrointestinal diseases such as inflammatory    bowel disease or ulcers, oral diseases such as dental caries, oral    cancer or periodontitis, ischemia reperfusion injury or sepsis;-   (xx) the antibody binds to CD32b and is for use in enhancement of    T-cell responses to tumor antigens and ADCC/phagocytosis by    macrophages, in combination with another therapeutic antibody;    vaccination, immunotherapy of B-cell lymphoma's, asthma or allergy;-   (xxi) the antibody binds to CD200 or CD200R and is for use in the    treatment of: asthma, rheumatoid arthritis, GVHD, other autoimmune    diseases, or cancer, such as solid tumors or lymphomas;-   (xxii) the antibody binds to Killer Immunoglobulin-Like Receptors    (KIRs), NKG2D or related molecules, leukocyte-associated    immunoglobulin-like receptors (LAIRs), or ly49 and is for use in the    treatment of: cancer, such as solid tumors or lymphomas, asthma,    rheumatoid arthritis, GVHD or other autoimmune diseases;-   (xxiii) the antibody binds to PD-L2 and is for use in the treatment    of cancer, asthma, or for use in vaccine enhancement;-   (xxiv) the antibody binds to CD26 and is for use in the treatment    of: atherosclerosis, GVHD, or auto-immune diseases;-   (xxv) the antibody binds to BST-2 and is for use in the treatment of    asthma, atherosclerosis, rheumatoid arthritis, psoriasis. Crohn's    disease, ulcerative cholitis, atopic dermatitis, sepsis or    inflammation;-   (xxvi) the antibody binds to ML-IAP (melanoma inhibitor of apoptosis    protein) and is for use in the treatment of melanoma,-   (xxvii) the antibody binds to cathepsin D and is for use in the    treatment of malignant diseases such as breast cancer, ovarian    cancer, oliorna, NSCLC, bladder cancer, endometrial cancer, liver    cancer, sarcoma, gastric cancer, SCCHN, prostate cancer or    colorectal cancer;-   (xxviii) the antibody binds to CD40 or CD40R and is for use in the    treatment of cancer, in particular B-cell lymphomas, B-cell-related    or -mediated diseases, autoimmune diseases such as psoriatic    arthritis, rheumatoid arthritis, multiple sclerosis, psoriasis,    Crohn's disease or ulcerative cholitis;-   (xxix) the antibody binds to CD86 and is for use in conjunction with    organ transplantation;-   (xxx) the antibody binds to a B cell receptor and is for use in the    treatment of: B-cell-related or -mediated diseases, such as B cell    lymphoma's, leukemia, autoimmune diseases, inflammation or allergy;-   (xxxi) the antibody binds to CD79 and is for use in the treatment of    B-cell-related or -mediated diseases, such as B-cell lymphomas,    leukemia, autoimmune diseases, inflammation or allergy;-   (xxxii) the antibody binds to a T cell receptor and is for use in    the treatment of T-cell-related or-mediated diseases, such as T-cell    lymphomas, leukemia, autoimmune diseases, inflammation or allergy;-   (xxxiii) the antibody binds to FcalphaR1 and is for use in the    treatment of a disease or disorder selected from allergic asthma or    other allergic diseases such as allergic rhinitis,    seasonallperennial allergies, hay fever, nasal allergies, atopic    dermatitis, eczema, hives, urticaria, contact allergies, allergic    conjunctivitis, ocular allergies, food and drug allergies, latex    allergies, or insect allergies, or IgA nephropathy, such as IgA    pemphigus;-   (xxxiv) the antibody binds to CD25 and is for use in the treatment    of a disease or disorder selected from the group consisting of    transplant rejection, graft-versus-host disease, inflammatory,    immune or autoimmune diseases, inflammatory or hyperproliferative    skin disorders, lymphoid neoplasms, malignancies, hematological    disorders, skin disorders, hepato-gastrointestinal disorders,    cardiac disorders, vascular disorders, renal disorders, pulmonary    disorders, neurological disorders, connective tissue disorders,    endocrinological disorders, and viral infections;-   (xxxv) the antibody binds to IL-15 or the IL15 receptor and is for    use in the treatment of a disease or disorder selected from the    group consisting of: arthritides, gout, connective disorders,    neurological disorders, gastrointestinal disorders, hepatic    disorders, allergic disorders, hematologic disorders, skin    disorders, pulmonary disorders, malignant disorders,    endocrinological disorders, vascular disorders, infectious    disorders, kidney disorders, cardiac disorders, circulatory    disorders, metabolic disorders, hone, disorders and muscle    disorders;-   (xxxvi) the antibody binds to IL-8 and is for use in the treatment    of a disease or disorder selected from the group consisting of    paimoplantar pustulosis (PPP), psoriasis, or other skin diseases,    inflammatory, autoimmune and immune disorders, alcoholic hepatitis    and acute pancreatitis, diseases involving 1L-8 mediated    angiogenesis;-   (xxxvii) the antibody binds to CD20 and is for use in the treatment    of a disease or disorder selected from the group consisting of:    rheumatoid arthritis, (autojimmurre and inflammatory disorders,    non-Hodgkin's lymphoma, B-CLL, lymphoid neoplasms, malignancies and    hematological disorders, infectious diseases and connective    disorders, neurological disorders, gastrointestinal disorders,    hepatic disorders, allergic disorders, hematologic disorders, skin    disorders, pulmonary disorders, malignant disorders,    endocrinological disorders, vascular disorders, infectious    disorders, kidney disorders, cardiac disorders, circulatory    disorders, metabolic disorders, bone and muscle disorders, and    immune mediated cytopenia;-   (xxxviii) the antibody binds to CD38 and is for use in the treatment    of a disease or disorder selected from the group consisting of    tumorigenic disorders, immune disorders in which CD38 expressing B    cells, plasma cells, monocytes and T cells are involved, acute    respiratory distress syndrome and choreoretinitis, rheumatoid    arthritis, inflammatory, immune and/or autoimmune disorders in which    autoantibodies and/or excessive B and T lymphocyte activity are    prominent, skin disorders, immune-mediated cytopenias, connective    tissue disorders, arthritides, hematologic disorders,    endocrinopathies, hepato-gastrointestinal disorders, nephropathies,    neurological disorders, cardiac and pulmonary disorders, allergic    disorders, ophthalmologic disorders, infectious diseases,    gynecological-obstetrical disorders, male reproductive disorders,    transplantation-derived disorders;-   (xxxix) the antibody binds to EGFr and is for use in the treatment    of a disease or disorder selected from the group consisting of:    cancers (over)expressing EGFr and other EGFr related diseases, such    as autoimmune diseases, psoriasis, and inflammatory arthritis;-   (xxxx) the antibody binds to CD4 and is for use in the treatment of    a disease or disorder selected from the group consisting of    rheumatoid arthritis, (auto)immune and inflammatory disorders,    cutaneous T cell lymphomas, non-cutaneous T cell lymphomas, lymphoid    neoplasms, malignancies and hematological disorders, infectious    diseases, and connective disorders, neurological disorders,    gastrointestinal disorders, hepatic disorders, allergic disorders,    hematologic disorders, skin disorders, pulmonary disorders,    malignant disorders, endocrinological disorders, vascular disorders,    infectious disorders, kidney disorders, cardiac disorders,    circulatory disorders, metabolic disorders, bone disorders, muscle    disorders, immune mediated cytopenia, and HIV infection/AIDS;-   (xxxxi) the antibody binds CD28 and is for use in the treatment of a    disease or disorder selected from the group consisting of an    inflammatory disease, autoimmune disease and immune disorder;-   (xxxxii) the antibody binds to tissue factor, or a complex of Factor    VII and tissue factor and is for use in the treatment of a disease    or disorder selected from the group consisting of vascular diseases,    such as myocardial vascular disease, cerebral vascular disease,    retinopathy and macular degeneration, and inflammatory disorders; or-   (xxxxiii) the antibody binds to PD-1 and is for use in the treatment    of HIV-1/AIDS.

In a further embodiment the invention relates to a pharmaceuticalcomposition, characterized in that it comprises a stabilized IgG4antibody as defined in any one of the above embodiments and apharmaceutically acceptable carrier or excipient.

In a further embodiment the invention relates to the use of a stabilizedIgG4 antibody according to any one of the above embodiments (i) to(xxxxiii) for the preparation of a medicament for the treatment of adisease as specified in any one of the above related embodiments (i) to(xxxxiii).

In a further embodiment the invention relates to a method for thetreatment of a subject suffering from a disease as specified in any oneof the above embodiments (i) to (xxxxiii) comprising administering tothe subject in need thereof a stabilized IgG4 antibody according to asspecified in any one of the above related embodiments (i) to (xxxxiii).

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting.

EXAMPLES Example 1 Structural Analysis of CH3-CH-3 Interface

In human IgG1, the non-covalent interaction between the CH3 domainsinvolves 16 residues located on four anti-parallel 13-strands that makeintermolecular contacts and burry 1090^(Å2) from each surface(Deisenhofer, J.; Biochemistry, 1981. 20(9): p. 2361-70). Alaninescanning mutagenesis showed that stabilization of the IgG1 CH3-CH3interaction was largely mediated by 6 of these residues, including K409(Dall′Acqua, W., et al.; Biochemistry, 1998. 37(26): p. 9266-73). To geta better understanding of the role of K409 in the IgG1 CH3-CH3interaction, the 1.65 Å1L6X crystal structure (Idusogie, E. E., et al.;Immunol, 2000. 164(8): p. 4178-84) was studied in more detail using theBrugel modelling package (Delhaise, P., et al., J. Mol. Graph., 1984.2(4): p. 103-106).

In order to propose mutations that should lead to a desiredstabilization (or destabilization) of IgG4, a quantitativestructure-based scoring methodology was employed (Desmet, J., et al.,Proteins, 2005. 58(1): p. 53-69). Briefly, each position in the CF3-CH3dimer interface was subjected to mutagenesis to all natural amino acids,except cysteine and praline. Subsequent to mutagenesis, Exploration ofthe conformational space was obtained by interdependent optimization ofthe side chains of all residues located in a sphere of 12 A of themutated residue, using the FASTER algorithm (Desmet, J., et al.,Proteins, 2002. 48(1): p. 31-43), performed on all macro-rotamericstates for the side chain under investigation. Subsequently, on eachmacro-rotameric state thus obtained, a scoring function for the sidechain under investigation was evaluated, as described (Desmet, J., etal.; Proteins, 2005. 58(1); p. 53-69). Finally, per position in theCH3-CH3 dimer interface, the highest scores for each mutation werecompared, and visual inspection of the resulting conformation wascarried out in selected cases,

Example 2 Water Hypothesis

In the IgG1 structure, K409 forms a hydrogen bond with D399′ on theopposite CH3 domain. Furthermore, K409 is part of a water-binding pockettogether with S364 and T411 in the same CH3 domain and K370′ on theopposite CH3 domain. The presence of the water molecule prevents anelectrostatic clash between K409 and K370′.

The K409R substitution (as in IgG4) was modelled in the 1L6X structureby optimizing the side chain conformations of the arginine residue andits surrounding residues, using the FASTER algorithm (Desmet, J., etal., Proteins, 2002. 48(1): p. 31-43). In this model, the guanidiniumgroup of R409 takes up the position of the water molecule and causes anelectrostatic clash with K370′. The side-chains of T411 and K370′ loosetheir interactions compared to the case with water present (as in IgG1),but D399 keeps its interaction with the side chain at position R409.

Example 3 Destabilization of IgG4

The mutations in the Table below were made in order to destabilize theCH3-CH3 interaction of an IgG4.

KABAT indicates amino acid numbering according to Kabat (Kabat et al.,Sequences of Proteins of immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md., (1991). EU indexindicates amino acid numbering according to EU index as outlined inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed,Public Health Service, National Institutes of Health, Bethesda, Md.(1991)).

Numbering of CH3 mutations KABAT EU index G4 SEQ ID NO: 4 370 Y349R*Y217R* 372 L351N* L219N* 372 L351Q* L219Q* 378 E357A E225A 378 E357T*E225T* 378 E357V* E225V* 378 E357I* E225I* 387 S364R* S232R* 387 S364K*S232K* 389 T366A T234A 389 T366R* T234R* 389 T366K* T234K* 389 T366N*T234N* 391 L368A L236A 391 L368V L236V 391 L368E* L236E* 391 L368G*L236G* 391 L368S* L236S* 391 L368T* L236T* 393 K370A K238A 393 K370R*K238R* 393 K370T K238T 427 D399A D267A 427 D399T* D267T* 427 D399S*D267S* 436 F405A F273A 436 F405L F273L 436 F405T* F273T* 436 F405D*F273D* 436 F405R* F273R* 436 F405Q* F273Q* 436 F405K* F273K* 436 F405YF273Y 438 Y407A Y275A 438 Y407E* Y275E* 438 Y407Q* Y275Q* 438 Y407K*Y275K* 438 Y407F Y275F 440 R409A R277A 440 R409K R277K (stabilizing seeWO2008145142) 440 R409E* R277E* 442 T411D* T279D* 442 T411V* T279V* 442T411N* T279N*

Example 4 Various Technical Procedures

The following techniques were performed as described in WO2007059782:Oligonucleotide primers and FOR amplification, agarose gelelectrophoresis, analysis and purification of PCR products and enzymaticdigestion products, quantification of DNA by UV spectroscopy,restriction enzyme digestions, ligation of DNA fragments, transformationof E. coli, screening of bacterial colonies by PCR, plasmid DNAisolation from E. coli culture, site-directed mutagenesis, DNAsequencing and transient expression in HEK-293F cells,

Example 5 Construction and Biochemical Analysis of CH3 Variants of2F8-HG

The above-described mutations were introduced into the CH3 region ofhingeless anti-EGFR antibody 2F8-HG, described in WO200/059782. To makethe constructs for the expression of the CH3 mutants, the mutations wereintroduced into pTomG42F8HG (described in WO2007059782) usingsite-directed mutagenesis. The constructs were expressed transiently andpurified as described in WO2007059782.

In order to investigate whether CH3 variant HG molecules exist asmonomers or dimers, a mass spectrometry method was employed as describedin WO2007059782.

FIG. 1 shows a summary of the monomer/dimer ratios obtained for each HGmutant using non-covalent nano-electrospray mass spectrometry. Ch3mutants showed a substantial increase in monomer/dimer ratio compared to2F8-HG (WT). The percentage molecules present as monomers increased from15% in 2F8-HG (WT) to >80% in most CH3 mutants, except for mutationR277A. HG mutation R277K, which introduces an IgG1 sequence into theIgGG4 backbone, was used as negative control. As expected, this mutantbehaved as dimer.

The monomer or dimer configuration of CH3 mutants was verified usingNativePAGE™ Novex® Bis-Tris gel electrophoresis (lnvitrogen, Carlsbad,Calif.) according to the instructions of the manufacturer as shown inFIG. 2. This native gel electrophoresis technique uses Coornassie G-250as a charge-shift molecule instead of SDS and is able to maintain nativeprotein conformation and protein complex quaternary structures (SchaggerH and von Jagow G 1991 Blue native gel electrophoresis for isolation ofmembrane complexes in enzymatically active form. Anal. Biocheim199:223-244).

Under these experimental conditions, 2F8-HG (WT) and R277K and R277Ashowed a protein band corresponding to the size of a full tetrameric(two heavy and two light chains) molecule. The CH3 mutants T234A, L236A,L236V, F273A, F2731L, and Y275A were shown to be half molecules (onlyone heavy and one light chain).

Example 6 Functional Analysis of CH3 Mutants of 2F8-HG

Binding of 21F8-HG (WT) and variants was determined in the absence andpresence of 200 μg/ml polyclonal human IgG (Intravenous Immunoglobulin,IVIG, Sanquin Netherlands) (as described in Example 57 of WO2007059782).

FIGS. 3 and 4 show that the binding curve of 2F8-HG in the presence ofIVIG clearly right-shifts with respect to the binding curve of 2F8-HGwithout IVIG. This difference in avidity for the EGFr coat is consistentwith the idea that, in the presence of IVIG, 2F8-HG binds monovalently(see Example 57 of WO2007059782). The binding curves of several of thetested mutations, 2F8-HG-T234A, 2F8-HG-L236V, 2F8HG-L236A and2F8-HG-Y275A, become insensitive to the addition of IVIG and weresuper-imposable ore the monovalent binding curve of 2F8-HG in thepresence of IVIG. These differences in avidity for the EGFr coat areconsistent with the idea that the 2F8-HG-T234A, 2F8-HG-L236V,2F8-HG-L236A and 2F8-HG-Y275A mutations prevent dimerization of the HGmolecules.

Example 7 Functional Analyses of CH3 Mutants of 2F8-HG

CH3 mutants of 2F8-HG were shown to bind EGFr with lower apparentaffinities than 2F8-HG in a binding ELISA coated with EGFr protein (seeabove). The potency of 2F8-HG CH3 mutants to inhibit ligand-induced EGFrphosphorylation in cells in vitro was compared to that of 2F8-HG (WT)and 2F8-Fab fragments in the Phosphorylation Inhibition Assay (PIA) asdescribed in example 54 of WO2007059782.

CH3 HG mutants were less potent to inhibit EGFr phosphorylation than2F8-HG (WT) and the control mutants R277K and R277A, in line with theincrease in monomer/dimer ratio of these mutants (FIG. 5).

Example 8 Concentration Dependent Configuration of CH3 Mutants of HG

The monomer/dimer configuration of CH3 mutants F273A, L236V, and Y275Awas further investigated at different concentrations, ranging from0.01-10 μM using non-covalent nano-electrospray mass spectrometry asdescribed in WO2007059732. The monomer/dimer configuration of these CH3mutants was compared to the configuration of 2F8-HG (WT) and R277K.

FIG. 6 shows that all HG mutants were 100% monomeric at lowconcentrations (except for R277K which behaved as dimer). With increasedconcentration of HG mutants, a decrease in monomericity was observed.However, the figure shows that the CH3 mutants exhibited such decreasein monomericity at much higher concentration than 2F8-HG (WT). Hence,the CH3 mutants contained a higher percentage of monomer molecules athigher molar concentrations.

For 2F8-HG (WT) and mutants E225A, E225V, S232R, T234A, L236A, L236T,L236V, L236E, L236S, L236G, K238A, K238T, D267S, D267A, F273A, F273L,F273Y, F273D, F273T, F273R, F273Q, Y275A, Y275Q, Y275K, Y275E, R277A,R277K, D267S+Y275E, DÐS+Y275Q, F273D+Y275E and F273T+Y275E signalscorresponding to the monomeric (M_(s),) and dimeric (D_(s))configurations were integrated and the relative proportion of eachconfiguration at each concentration ([M]₀) was determined using thefollowing equations:

[M] _(eq) =M _(S)/(M _(S) +D _(S))·[M] ₀: concentration monomer atequilibrium

[D] _(eq) =([M] ₀ [M] _(eq))/2; concentration dimer at equilibrium

Dissociation constant (K_(D)) values were subsequently calculated forall mutants by plotting the [D]_(eq) against [M]_(eq) ² values of eachconcentration and determining the gradient by least-squares linearregression using Excell software (Microsoft), The K_(D) measured for2F8-HG (WT) was 5.0×10 ⁻⁸ M. The relative K_(D) of each mutant comparedto the K_(D) of 2F8-HG (WT) was calculated and plotted.

FIG. 7 shows that all HG mutants (except for R277K. K238A and K238T) hada higher relative K_(D), which translates into an increase in monomericbehavior compared to 2F8-HG (WT). The R277K, K238A and K238Tmutantsshowed a lower relative K_(D), meaning that they stabilize the CH3-CH3interaction.

Example 9 Removal of Glycosylation Sites

To remove (potential) acceptor sites for N-linked glycosylation(“glycosylation sites”) from the monovalent antibody, alterations to thesequence were made. To examine how this could be achieved withintroducing a minimum of T cell epitopes, and without perturbing thenative structure of the molecule, an in silico analysis was performed.The HLA binding specificities of all possible 10-mer peptides derivedfrom a target sequence were analyzed (Desmet et al. 1992, 1997, 2002,2005; Van Walle et al. 2007 Expert Opinion on Biological Therapy7:405-418). Profiling was done at the allotype level for 20 DRB1, 7DRB3/4/5, 14 DQ and 7 DP, i.e. 48 HLA class ll receptors in total.Quantitative estimates of the free energy of binding deltaGbind of apeptide for each of the 48 HLA class II receptors were calculated. Thesedata were then further processed by classifying peptides as strong,medium, weak and non-binders.

The table below shows the 27 sequence variants which contain only mediumepitopes, specific for no more than three different DRB1 allotypes.

TABLE Summary of sequence variants containing either a single mediumDRB1 epitope, or multiple medium epitopes affecting three or less MHCallotypes. The first column contains the specific sequence, the secondcolumn the number of medium DRB1 binding epitopes present in thesequence fragment, and the subsequent columns describe the specificityof these epitopes. Allotypes for which no epitopes were found in any ofthese sequence fragments were not included in the table. DRB1M DRB1*0101DRB1*0102 DRB1*0401 DRB1*0402 DRB1*0405 DRB1*0407 DRB1*0801 NST 0 DST 11 1 EST 1 1 1 GST 1 1 HST 1 1 1 MST 1 1 PST 1 1 QST 1 1 1 1 SST 1 1 1TST 1 1 1 CSE 2 1 1 CSP 2 1 1 DSE 2 1 1 DSG 2 1 1 DSP 2 1 1 ESE 2 1 1 1ESP 2 1 1 GSE 2 1 1 1 GSP 2 1 1 HSE 2 1 1 MSE 2 1 1 1 NSE 2 1 1 NSP 2 11 PSE 2 1 1 1 PSP 2 1 1 SSE 2 1 1 SSP 3 1 1 1 TSP 3 1 1 1 DRB1*0802DRB1*0901 DRB1*1101 DRB1*1104 DRB1*1301 DRB1*1401 NST DST 1 1 EST 1 GST1 HST 1 1 MST 1 PST 1 1 QST 1 1 1 1 1 SST 1 TST 1 1 1 CSE CSP 1 DSE DSG1 DSP ESE ESP GSE GSP HSE MSE NSE NSP 1 PSE PSP SSE SSP TSP

The lowest epitope content found in the study was within sequencevariants which bind with medium strength to two different DRB1 allotypes(GST, MST, CSE, DSE, DSP, ESP, GSP, HSE, NSE, PSP and SSE). A negativeselection for mutations that:

substitute any positions to cysteine,

change the final threonine to proline, or

replace the initial asparagines residue by an aliphatic side chain, leadto the selection of the following preferred candidates: GST, NSE, DSE,HSE and SSE.

To make the constructs for the expression of deglycosylated 2F8-HG, theGST and NSE mutations as identified by the above-described analysis wereintroduced into pTomG42F8HG (described in WO 2007059782) usingsite-directed mutagenesis. The constructs were expressed transiently andbinding was determined in the absence and presence of polyclonal humanIgG (Intravenous Immunoglobulin, IVIG, Sanquin Netherlands) (asdescribed in Example 57 of WO 2007059782).

FIG. 8 shows that the binding curves of 2F8-HG-GST and 2F8-HG-NSE in theabsence and presence of IVIG were identical to the binding curve of2F8-HG in the absence and presence of IVIG, respectively. This isconsistent with the hypothesis that deglycosylation does not effect thebinding affinity of the HG-molecules or sensitivity to IVIG.

Example 10 Biochemical Analysis of Non-Glycosylation Mutants of 2F8-HG

Absence of glycosylation in the glycosylation site mutants of 2F8-HG wasconfirmed using High pH Anion Exchange Chromatography-Pulse AmperometricDetection (HFAEC-PAD).

To investigate the monomeric or dimeric configuration of the mutated HGmolecules, a specialized mass spectrometry method was employed topreserve non covalent interactions between molecules.

HG mutant samples were prepared in aqueous 50 mM ammonium acetatesolutions and introduced into an LC-T nanoelectrospray ionizationorthogonal time-of-flight mass spectrometer (Micromass, Manchester, UK),operating in positive ion mode. Source pressure conditions in the LC-Tmass spectrometer and nano-electrospray voltages were optimized foroptimal transmission, the pressure in the interface region was adjustedby reducing the pumping capacity of the rotary pump by closing the valve(Pirani Pressure 6.67e0 mbar).

Spraying conditions were .as follows: needle voltage 1275 V, conevoltage 200 V, and source temperature 80° C. Borosilicate glasscapillaries (Kwik-Fil™, World Precision instruments Inc., Sarasota,Fla.) were used on a P-97 puller (Sutter Instrument Co., Novato, Calif.)to prepare the nano-electrospray needles. They were subsequently coatedwith a thin gold layer using an Edwards Scancoat six Pirani 501 sputtercoater (Edwards High Vacuum International, Crawley, UK).

FIG. 9 shows a summary of the monomer/dimer ratios obtained for each HGmutant using non-covalent nano-electrospray mass spectrometry at 1 μMprotein concentrations. In agreement with the observations described inExample 54 of WO2007059782, the data indicate that in the absence ofpolyclonal human IgG, 2F8-HG may behave as a bivalent antibody.

Under these experimental conditions, non-glycosylation mutants exhibitedthe same monomer/dimer ratio as 2F8-HG (WT).

Example 11 Functional Analysis of Non-Glycosvlation Mutants of 2F8-HG

Non-glycosylation HG mutants 2F8-HG-GST, 2F8-HG-NSE, 2F8-HG-DSE,2F8-HG-HSE, and 2F8-HG-SSE were shown to bind EGFr with apparentaffinities similar to 2F8-HG (WT) in a binding ELISA, using EGFr proteinas coat (see above). The potency of non-glycosylation 2F8-HG mutants toinhibit ligand-induced EGFr phosphorylation in cells in vitro wascompared to that of 2F8-HG (WT) and 2F8-Fab fragments in thePhosphorylation Inhibition Assay (PIA) as described in example 54 ofWO2007059782. FIG. 10 shows that the potency of non-glycosylation HGmutants to inhibit EGF-induced phosphorylation of EGFr in vitro wassimilar to that of 2F8-HG (WT).

Example 12 Pharmacokinetic Evaluation of Non-Glycoasylation Mutants

Pharmacokinetic characteristics of non-glycosylation mutant 2F8-HG-GSTand 2F8-HG-NSE were analyzed in SCID mice supplemented with 0.1 mg7D8-IgG1 as internal control. Pharmacokinetic analysis is explained indetail in example 50 of WO2007059782, Internal control 7D8-IgG1exhibited an equal clearance rate in at mice investigated and wascomparable to the clearance rate of 2F8-IgG4.

FIG. 11 shows that absence of glycosylation of 2F8-HG did not affectplasma clearance.

Example 13

Generation of IgG1 and IgG4 Antibodies with Hinge Region and/or CH3Domain Mutations

To investigate the structural requirements for Fab arm exchange, fiveIgG1 mutants were made: an IgG1 with an IgG4 core-hinge (IgG1-P228S)(corresponds to 111 in SEQ ID NO:7), two CH3 domain swap mutants(IgG1-CH3(γ4) and IgG1-P228S-CH3(γ4)), one CH3 point mutant in whichlysine present at position 409 of IgG1 (within the CH3 domain)(corresponds to 292 in SEQ ID NO:7) is replaced for arginine(IgG1-K409R), and one IgG1 with an IgG4 core hinge and K409F1 mutation(IgG1-P228S-K409R) (FIG. 12). These mutants were made with either Bet v1or Fel d1 specificity. Please see WO 2008/119353 (Genmab A(S),especially the examples, for a further description of production ofantibody mutants as well as the Bet v1 and Fel d1 specificities.

Two IgG4 mutants were made: one CH3 point mutant in which argininepresent at position 409 of IgG4 (within the CH3 domain) (corresponds to289 in SEQ ID NO:2) is replaced for lysine (IgG4-R409K), and one CH3swap mutant (1g04-CH3(γ1)) (FIG. 12). These mutants were also made witheither Bet v1 or Fel d1 specificity.

Site directed mutagenesis was used to introduce a P228S mutation in thehinge of IgG1 using pEE-G1-wt a Bet v1 as a template. Quickchangesite-directed mutagenesis kit (Stratagene) was used to create thepEE-G1-CFSC mutant. The polymerase chain reaction (PCR) mix consisted of5 μl pEE-G1 a Betv1 DNA template (˜35 ng), 1.5 μl mutagenicprimer-forward (˜150 ng), 1.5 μl mutagenic primer-reverse (˜150 ng), 1μl dNTP mix, 5 μl reaction buffer (10×), 36 μl H2O and finally 1 μl PluTurbo DNA polymerase. Then the mix was applied to the PCR: 30″95° C.,30″95° C. (denaturating), 1′55° C. (annealing) and 17 minutes 68° C.(elongating). This cycle was repeated 20 times.

DNA digesting and ligation was used to create CH3 domain swap mutantconstructs IgG1 CH3(y4) and IgG1-P2288-CH3(y4). Digestion reactions toobtain CH3 domains and vectors without CH3 domains were as follows:˜1500 ng DNA (pEE-G1-betv1, pEE-G1-CPSC and pEE-G4-betv1), 2 μl BSA, 2μl Neb3 buffer, 1 μl Sail and H₂O added to a volume of 20 μl. Incubationat 37° C. for 30′. DNA was purified and eluted with 30 μl H₂O before 1μl San Dl and 3 μl universal buffer was added and incubated at 37° C.for 30′. Fragments were subjected to gel electrophoresis on 1% agarosegels with ethidium bromide. Fragments were cut from the gel underultraviolet light and dissolved using a DNA purification kit (Amersham).The pEE-G4-wt Sall/SanDl (which contained IgG4 CH3 domain) fragment wasligated into pEE-G1-wt and pEE-G1-CPSC using following procedure: 1 μltemplate DNA (SaII/SanDI)I digested pEE-G1-wt and pEE-G1-CPSC), 5 μlSaII/SanDI insert, 4 μl Ligate-it buffer, 9 μl H₂O and 1 μl ligase in atotal volume of 20 μl. Ligation was stopped after 5′.

DNA digestion (using ApaI and HindIII) and ligation was used to replacethe VH domain of the bet v1 mutant antibodies with that ofpEE-G4-a-feld1 wt, following a similar procedure as above.

Site-directed mutagenesis was used to introduce point mutations (K409Ror R409K) into the pEE-γ4 wt, pEE-γ1 and PEE-γ1-P228S constructs.Site-directed mutagenesis was performed using the QuickChange II XLSite-Directed Mutagenesis Kit (Stratagene, Amsterdam, The Netherlands)according to the manufacturer's instructions, with changes as indicatedbelow to increase mutagenic efficiency. This method included theintroduction of a silent extra Reel site to screen for successfulmutagenesis. First, a prePCR mix was used containing 3 μl 10× pfureaction buffer, 1 μl dNTP mix (10 mM), 275 ng forward or reverseprimer, 50 ng template DNA and 0.75 μl Flu turbo hotstart polymerase. AprePCR was run using a GeneAmp PCR system 9700 (Applied Biosystems):initial denaturation at 94° C. for 5 min; 4 cycles of 94° C. for 30 sec,50° C. for 1 rain and 68° C. for 14 min, 25 μl of forward primercontaining prePCR mix was added to 25 μl of reverse primer containingprePCR mix. 0.5 μl Pfu turbo hotstart was added and amplification wasperformed: denaturing at 94° C. for 1 min; 14 cycles of 94° C. for 1min, 50° C. for 1 min and 68° C. for 8 min; 12 cycles of 94° C. for 30sec, 55° C. for 1 min and 68° C. for 8 min.

PCR mixtures were stored at 4° C. until further processing. Next, PCRmixtures were incubated with 1 μl DpnI for 60 min at 37° C. and storedat 4° G until further processing. 2 μl of the digested PCR products wastransformed in One Shot DNH5α T1^(A) competent E. coil cells(Invitrogen, Breda, The Netherlands) according to the manufacturer'sinstructions (Invitrogen). Next, cells were plated on Luria-Bertani (LB)agar plates containing 50 μg/ml ampicillin. Plates were incubated for16-18 hours at 37° C. until bacterial colonies became evident. Afterscreening by colony PCR and Accl digestion to check for successfulmutagenesis, plasmid was isolated from the bacteria and the mutation wasconfirmed by DNA sequencing. To check if no unwanted extra mutationswere introduced the whole HC coding region was sequenced and did notcontain any additional mutations.

Recombinant antibodies from these constructs were transiently expressedin HEK 293 cells in 3 ml, 6-wells plates (NUNC) or in 125 or 250erienmeyers (Corning) with 293 Pectin (Invitrogen) as transfectionreagent.

Example 14 Pab Arm Exchange of IgG1 and IgG4 Hinge Region or CH3 DomainMutants

Antibodies were mixed and subsequently incubated with reducedglutathione (GSH) to investigate the exchange of half molecules. GSH(Sigma-Aldrich, St. Louis, Mo.) was dissolved in water before use.

The exchange of half molecules was evaluated by incubating an antibodymixture consisting of Bet v1 specific antibody (200 ng) and Fel d1specific antibody (200 ng) in PBS/Azide containing GSH (1 or 10 mM) at37° C. Total incubation volume was 50 μl. After 24 hours samples weredrawn from the incubation mixture in PBS-AT (PBS supplemented with 0.3%bovine serum albumin, 0.1% Tween-20 and 0.05% (w/v) NaN₃). For samplescontaining 10 mM GSH an equimolar amount of iodine-acetamide, a stronglyalkyiating agent that inhibits the GSH activity, was added. Samples werestored at 4° C. for measuring of antigen binding and bispecific activity

Levels of Bet v1 binding antibodies were measured in the antigen bindingtest. Samples were incubated with 0.75 mg of protein G Sepharose(Amersham Biosciences, Uppsala, Sweden) in 750 μl PBS-IAT (PBS-ATsupplemented with 1 μg/ml IVIg) in the presence of ¹²⁵ l -labeled Bet v1for 24 h. Next, the Sepharose was washed with PBS-T (PBS supplementedwith 0.1% Tween-20 and 0.05% (w/v) NaN₃) and the amount of radioactivitybound relative to the amount of radioactivity added was measured. Theconcentration of Bet v1 specific IgG was calculated using purified Betv1 specific antibodies as a standard (range 0-200 ng per test asdetermined by nephelometer).

The concentration of bispecific IgG (Le. Fel d1-Bet v1 cross-linkingactivity) was measured in the heterologous cross-linking assay. In thisassay, a sample was incubated for 24 h with 0.5 mg Sepharose-coupled catextract, in which Fel d1 antigen is present, in a total volume of 300 μlin PBS-IAT. Subsequently, the Sepharose was washed with PBS-T andincubated for 24 h with ¹²⁵I-labeled Bet v1, after which the Sepharosewas washed with PBS-T and the amount of radioactivity bound relative tothe amount of radioactivity added was measured. The concentration ofbispecific IgG (Fel d1-Bet v1) was calculated using the same calibrationcurve as used in the Bet v1 binding test, which was obtained frompurified Bet v1 binding IgG. Tests were performed usingantibody-containing supernatants in FreeStyle 293 expression medium,GIBCO/invitrogen Corporation.

The following antibody mixtures were used:

-   Betv1-IgG1 wt with Feld1-IgG1 wt (indicated as IgG1 wt in FIG. 13)-   Betv1-IgG1 P228S with Feld1-IgG1-P228S in FIG. 13)-   Betv1-IgG4-CH3(y1) with Feld1-IgG4-CH3(γ1) (IgG4-CH3(γ1) in FIG. 13)-   Betv1-IgG4-R409K with Feld1-IgG4-R409K (IgG4-R409K in FIG. 13)-   Betv1-IgG1-CH3(γ4) with Feld1-IgG1-CH3(γ4) (IgG1-CH3(y4) in FIG. 13)-   Betv1-IgG1-K409R with Feld1-IgG1-K409R (IgG1-K409R in FIG. 13)-   Betv1-IgG4 wt with Feld1-IgG4 wt (IgG4 wt in FIG. 13)-   Betv1-IgG1-P228S-CH3(y4) with Feld1-IgG1-P228S-CH3(γ4)    (IgG1-P228S-CH3(γ4) in FIG. 13)-   Betv1-IgG1-P228S-K409R with Feld1-IgG1-P228S-K409R (IgG1-P228S-409R    in FIG. 13)

The results (FIG. 13) showed that at 1 mM GSH, half molecule exchangeoccurs between lgG4 wt, IgG1-P228S-K409R or IgG1-P228S-CH3(γ4)antibodies. Under these conditions, IgG1 wt, IgG1-P228S, IgG4-CH3(γ1),IgG4-R409K, IgG1-CH3(γ4) or IgG1-K409R antibodies showed no or onlyminimal exchange of half molecules. At 10 μM GSH, half molecule exchangewas also seen in the reactions containing IgG1-CH3(γ4) or IgG1-K409Rantibodies.

Example 15 Additional CH3 Mutations to Stabilize Dimerization ofHingeless IgG4 Antibody Molecules in the Absence of IVIG.

Hingeless IgG4 antibody (HG) molecules form dimers by low affinitynon-covalent interactions, WO120071059782 describes that thisdimerization process can be inhibited by using HG IgG4 molecules in thepresence of an excess of irrelevant antibodies. WO/2007/059782 describesa hingeless IgG4 anti-EGFR antibody 2F8-HG.

Construction of pHG-2F8: A vector for the expression of the heavy chainof 2F8-HG: The heavy chain cDNA encoding region of 2F8-HG was codonoptimized and cloned in the pEE6,4 vector (Lonza Biologics, Slough, UK).The resulting vector was named pHG-2F8.

Construction of pKappa2F8: A vector for the production of the lightchain of 2F8 antibodies: The VL region encoding antibody 2F8 was codonoptimized and cloned in the pKappa2F2 vector (a vector encoding thecodon optimized cDNA region of antibody 2F2 (described in WO2004035607)in vector pEE12.4 (Lonza)), replacing the 2F2 VL region with the 2F8 VLregion. The resulting vector was named pKappa-2F8.

Hingeless IgG4 anti-EGFR antibody 2F8-HG has been described inWO12007/059782. The additional mutations given in the Table below wereintroduced into the CH3 region of hingeless IgG4 antibody 2F8-HG bysite-directed mutagenesis.

KABAT indicates amino acid numbering according to Kabat (Kabat et al.,Sequences of Proteins of immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991).

EU index indicates amino acid numbering according to EU, index asoutlined in Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National institutes of Health,Bethesda, Md. (1991)). See also FIG. 14 for comparison of numberingmethods.

Numbering of CH3 mutations KABAT EU index G4 SEQ ID NO: 2 436 F405AF285A 436 F405L F285L 440 R409A R289A 440 R409K R289K

To make the constructs for the expression of the CH3 mutants, themutations were introduced into pHG2F8 using site-directed mutagenesis.

The constructs were expressed transiently in HEK-293F cells bycotransfecting the heavy-chain-and light-chain-encoding plasmids andbinding to purified EGFr was determined in the absence and presence of200 μg/ml polyclonal human IgG (Intravenous Immunoglobulin, IVIG,Sanquin Netherlands).

Binding affinities were determined using an ELISA in which purified EGFr(Sigma, St Louis, Mo.) was coated to 96-well Microlon ELISA plates(Greiner, Germany), 50 ng/well. Plates were blocked with PBSsupplemented with 0.05% Tween 20 and 2% chicken serum. Subsequently,samples, serially diluted in a buffer containing 100 μg/ml polyclonalhuman IgG (Intravenous Immunoglobulin, IVIG, Sanquin Netherlands) wereadded and incubated for 1 h at room temperature (RT). Plates weresubsequently incubated with peroxidase-conjugated rabbit-anti-humankappa light chain (DAKO, Glostrup, Denmark) as detecting antibody anddeveloped with 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)(ALTS; Roche, Mannheim, Germany). Absorbance was measured in amicroplate reader (Biotek, Winooski, Vt.) at 405 nm.

FIG. 14 shows that the binding curve of 2F8-HG in the presence of IVIG(thick dotted line with closed boxes) clearly right-shifts with respectto the binding curve of 2F8-HG without IVIG (thick closed line with openboxes). This difference in avidity for the EGFr coat is consistent withthe idea that, in the presence of IVIG, 2F8-HG binds monovalently. Thebinding curves of the tested mutations, 2F8-HG-F405L, 2F8-HG-F405A,2F8-HG-R409A and 2F8-HG-R409KA, become insensitive to the addition ofIVIG and were super-imposable on the bivalent binding curve of 2F8-HG inthe absence of IVIG. These differences in avidity for the EGFr coat areconsistent with the idea that the 2F8-HG-F405L, 2F8-HG-F405A,2F8-HG-R409A and 2F8-HG-R409K mutations stabilize dimerization of the HGmolecules.

Example 16 Additional CH3 Domain Mutations to Stabilize Dimerization ofHuman Antibodies

Following the analysis described in Examples 1 and 2, it washypothesized that in human IgG 4, mutations relieving the electrostaticstrain between P409 and K370 (indicated with 4 in the table below) couldpossibly be used to stabilize IgG4 and prevent Fab-arm exchange.Mutations were introduced into the CH3 domains of IgG4-CD20 andIgG4-EGFr by site-directed mutagenesis.

Mutations as given in the Table below were introduced into the CH3domains of IgG4-CD20 and IgG4-EGFr by site-directed mutagenesis.

KABAT indicates amino acid numbering according to Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). EU indexindicates amino acid numbering according to EU index as outlined Kabatet al., Sequences of Proteins of immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991)).See also FIG. 15 for comparison of numbering methods.

Numbering of CH3 mutations KABAT EU index G4 SEQ ID NO: 2 370 Y349DY229D 372 L351K L231K 376 Q355R Q235R 378 E357T E237T 387 S364D S244D393 K370E K250E 393 K370Q K250Q 436 F405A F285A 436 F405L F285L 440R409A R289A 440 R409K R289K 440 R409L R289L 440 R409M R289M 440 R409TR289T 440 R409W R289W 442 T411V T291V 450 E419Q E299Q 476 L445P L325P

IgG1-CD20 and IgG1-EGFr, IgG4-CD20 and IgG4-EGFr, or IgG4-0H3mutant-CD20and IgG4-CH3mutant-EGFr were mixed and incubated with 0.5 mM GSH asdescribed above. Bispecific activity was determined as described inExample 33 of PCT application, WO 2008/119353 (Genmab A/S),

FIG. 16 shows that bispecific anti-EGFrICD20 antibodies were formed inmixtures of IgG4 antibodies as well as in mixtures of CH3 domain mutantsQ235R, E299Q, L325P, R289A and S244D. No bispecific activity wasmeasured in mixtures of CH3 domain mutants R289K, R289M, R289L, K250Eand K250Q, indicating that these mutations stabilized dimerization ofhuman IgG4 antibodies. For CH3 domain mutants L231K, Y229D, F285A,F285L, R289W and E237T diminished bispecific activity was measured. TheCH3 domain mutant T291V was unique in that it slowed down the exchangereaction, but reached the same level of exchange as wild-type IgG4 after24 hrs.

Example 17 Kp Measurements in CH2 CH3 Constructs Based on IgG4 and IgG4CH3 Mutants

In order to investigate the CH3-CH3 interaction strength of IgG4,his-tagged constructs were designed based on the Fc-domains human IgG4lacking the hinge region to prevent covalent inter-heavy chain disulfidebonds, his-CH2-CH3(G4). Subsequently, variants of these constructscontaining mutations in the CH3-CH3 interface listed below weregenerated by site-directed mutagenesis.

KABAT indicates amino acid numbering according to Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). EU indexindicates amino acid numbering according to EU index as outlined inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)). See also FIG. 15 for comparison of numbering methods.

Numbering of CH3 mutations KABAT EU index G4 SEQ ID NO: 2 370 Y349DY229D 372 L351K L231K 378 E357T E237T 387 S364D S244D 393 K370E K250E393 K370Q K250Q 436 F405A F285A 436 F405L F285L 440 R409A R289A 440R409K R289K 440 R409L R289L 440 R409M R289M 440 R409W R289W

The monomer/dimer configuration of the his-CH2-CH3(G4) and CH3 mutantswas investigated at different concentrations, ranging from 0.01-10 μMusing non-covalent nano-electrospray mass spectrometry as described inWO2007059782. For his-CH2-CH3(G4) and CH3 mutants signals correspondingto the monomeric (M_(s)) and dimeric (D_(s)) configurations wereintegrated and the relative proportion of each configuration at eachconcentration ([M]_(c)) was determined as described in example 8.

The K_(D) measured for his-CH2-CH3(G4) (WT) was 4.8×10⁻⁸ M. The relativeK_(D) of each mutant compared to the K_(D) of his-CH2-CH3(G4) (WT) wascalculated and plotted.

FIG. 17 shows that CH3 mutants K250E, K250Q, R289L, R289M and R289K hada lower relative K_(D), which translates into stabilization of theCH3-CH3 interaction compared to his-CH2-CH3(G4) (WT). The S244D mutanthad a K_(D). value, which was comparable to his-CH2-CH3(G4) (WT). TheCH3 mutants Y229D, L231K, E237T, F285A, F285L, R289A and R289W showed ahigher relative K_(D), meaning an increase in monomeric behaviorcompared to his-CH2-CH3(G4) (WT).

FIG. 18 shows the correlation between the K_(D) values of the CH3mutants in relation to the % of bispecific activity (after 24 hrscompared to WT IgG4). Mutants that are stabilized do not show bispecificactivity, indicating that Fab-arm exchange does not occur in thesemutants. Mutants that have K_(D) values comparable to WT. IgG4 behavesimilar in the generation of bispecific antibodies. FIG. 16 and FIG. 18together show that mutants that have a weaker CH3-CH3 interaction doform bispecific antibodies, but the amount of bispecific antibodies ismuch lower and are not stable over time.

SEQUENCE LISTINGThe nucleic acid sequence of the wildtype C_(H )region of human IgG4SEQ ID No: 1:    1GCTAGCACCA AGGQCCCATC CGTCTTCCCC CTGGCGCCCT GCTCCAGGAG   51CACCTCCGAG AGCACAGCCG CCCTGGGCTG CCTGGTCAAG GACTACTTCC   101CCGAACCGGT GACGGTGTCG TGGAACTCAG GCGCCCTGAC CAGCGGCGTG   151CACACCTTCC CGGCTGTCCT ACAGTCCTCA GGACTCTACT CCCTCAGCAG   201CGTGGTGACC GTGCCCTCCA GCAGCTTGGG CACGAAGACC TACACCTGCA   251ACGTAGATCA CAAGCCCAGC AACACCAAGG TGGACAAGAG AGTTGGTGAG   301AGGCCAGCAC AGGGAGGGAG GGTGTCTGCT GGAAGCCAGG CTCAGCCCTC   351CTGCCTGGAC GCACCCCGGC TGTGCAGCCC CAGCCCAGGG CAGCAAGGCA   401TGCCCCATCT GTCTCCTCAC CCGGAGGCCT CTGACCACCC CACTCATGCT   451CAGGGAGAGG GTCTTCTGGA TTTTTCCACC AGGCTCCGGG CAGCCACAGG   501CTGGATGCCC CTACCCCAGG CCCTGCGCAT ACAGGGGCAG GTGCTGCGCT   551CAGACCTGCC AAGAGCCATA TCCGGGAGGA CCCTGCCCCT GACCTAAGCC   601CACCCCAAAG GCCAAACTCT CCACTCCCTC AGCTCAGACA CCTTCTCTCC   651TCCCAGATCT GAGTAACTCC CAATCTTCTC TCTGCAGAGT CCAAATATGG   701TCCCCCATGC CCATCATGCC CAGGTAAGCC AACCCAGGCC TCGCCCTCCA   751GCTCAAGGCG GGACAGGTGC CCTAGAGTAG CCTGCATCCA GGGACAGGCC   801CCAGCCGGGT GCTGACGCAT CCACCTCCAT CTCTTCCTCA GCACCTGAGT   851TCCTGGGGGG ACCATCAGTC TTCCTGTTCC CCCCAAAACC CAAGGACACT   901CTCATGATCT CCCGGACCCC TGAGGTCACG TGCGTGGTGG TGGACGTGAG   951CCAGGAAGAC CCCGAGGTCC AGTTCAACTG GTACGTGGAT GGCGTGGAGG 1001TGCATAATGC CAAGACAAAG CCGCGGGAGG AGCAGTTCAA CAGCACGTAC 1051CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC TGAACGGCAA 1101GGAGTACAAG TGCAAGGTCT CCAACAAAGG CCTCCCGTCC TCCATCGAGA 1151AAACCATCTC CAAAGCCAAA GGTGGGACCC ACGGGGTGCG AGGGCCACAT 1201GGACAGAGGT CAGCTCGGCC CACCCTCTGC CCTGGGAGTG ACCGCTGTGC 1251CAACCTCTGT CCCTACAGGG CAGCCCCGAG AGCCACAGGT GTACACCCTG 1301CCCCCATCCC AGGAGGAGAT GACCAAGAAC CAGGTCAGCC TGACCTGCCT 1351GGTCAAAGGC TTCTACCCCA GCGACATCGC CGTGGAGTGG GAGAGCAATG 1401GGCAGCCGGA GAACAACTAC AAGACCACGC CTCCCGTGCT GGACTCCGAC 1451GGCTCCTTCT TCCTCTACAG CAGGCTAACC GTGGACAAGA GCAGGTGGCA 1501GGAGGGGAAT GTCTTCTCAT GCTCCGTGAT GCATGAGGCT CTGCACAACC 1551ACTACACACA GAAGAGCCTC TCCCTGTCTC TGGGTAAA The amino acid sequence of the wildtype C_(H )region of human IgG4SEQ ID No: 2:     1ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV   51

  101

  151 PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVX1H QDWLNGKEYK   201

  251

  301

wherein X1 at position 189 is Leu and X2 at position 289 is Arg, orwherein X1 at position 189 is Leu and X2 at position 289 is Lys, orwherein X1 at position 189 is Val and X2 at position 289 is Arg. The nucleic acid sequence encoding the C_(H )region of human IgG4(SEQ ID No: 1) mutated in positions 714 and 722 SEQ ID No: 3:     1GCTAGCACCA AGGGCCCATC CGTCTTCCCC CTGGCGCCCT GCTCCAGGAG   51CACCTCCGAG AGCACAGCCG CCCTGGGCTG CCTGGTCAAG GACTACTTCC   101CCGAACCGGT GACGGTGTCG TGGAACTCAG GCGCCCTGAC CAGCGGCGTG   151CACACCTTCC CGGCTGTCCT ACAGTCCTCA GGACTCTACT CCCTCAGCAG   201CGTGGTGACC GTGCCCTCCA GCAGCTTGGG CACGAAGACC TACACCTGCA   251ACGTAGATCA CAAGCCCAGC AACACCAAGG TGGACAAGAG AGTTGGTGAG   301AGGCCAGCAC AGGGAGGGAG GGTGTCTGCT GGAAGCCAGG CTCAGCCCTC   351CTGCCTGGAC GCACCCCGGC TGTGCAGCCC CAGCCCAGGG CAGCAAGGCA   401TGCCCCATCT GTCTCCTCAC CCGGAGGCCT CTGACCACCC CACTCATGCT   451CAGGGAGAGG GTCTTCTGGA TTTTTCCACC AGGCTCCGGG CAGCCACAGG   501CTGGATGCCC CTACCCCAGG CCCTGCGCAT ACAGGGGCAG GTGCTGCGCT   551CAGACCTGCC AAGAGCCATA TCCGGGAGGA CCCTGCCCCT GACCTAAGCC   601CACCCCAAAG GCCAAACTCT CCACTCCCTC AGCTCAGACA CCTTCTCTCC   651TCCCAGATCT GAGTAACTCC CAATCTTCTC TCTGCAGAGT CCAAATATGG   701TCCCCCATGC CCACCATGCC CGGGTAAGCC AACCCAGGCC TCGCCCTCCA   751GCTCAAGGCG GGACAGGTGC CCTAGAGTAG CCTGCATCCA GGGACAGGCC   801CCAGCCGGGT GCTGACGCAT CCACCTCCAT CTCTTCCTCA GCACCTGAGT   851TCCTGGGGGG ACCATCAGTC TTCCTGTTCC CCCCAAAACC CAAGGACACT   901CTCATGATCT CCCGGACCCC TGAGGTCACG TGCGTGGTGG TGGACGTGAG   951CCAGGAAGAC CCCGAGGTCC AGTTCAACTG GTACGTGGAT GGCGTGGAGG 1001TGCATAATGC CAAGACAAAG CCGCGGGAGG AGCAGTTCAA CAGCACGTAC 1051CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC TGAACGGCAA 1101GGAGTACAAG TGCAAGGTCT CCAACAAAGG CCTCCCGTCC TCCATCGAGA 1151AAACCATCTC CAAAGCCAAA GGTGGGACCC ACGGGGTGCG AGGGCCACAT 1201GGACAGAGGT CAGCTCGGCC CACCCTCTGC CCTGGGAGTG ACCGCTGTGC 1251CAACCTCTGT CCCTACAGGG CAGCCCCGAG AGCCACAGGT GTACACCCTG 1301CCCCCATCCC AGGAGGAGAT GACCAAGAAC CAGGTCAGCC TGACCTGCCT 1351GGTCAAAGGC TTCTACCCCA GCGACATCGC CGTGGAGTGG GAGAGCAATG 1401GGCAGCCGGA GAACAACTAC AAGACCACGC CTCCCGTGCT GGACTCCGAC 1451GGCTCCTTCT TCCTCTACAG CAGGCTAACC GTGGACAAGA GCAGGTGGCA 1501GGAGGGGAAT GTCTTCTCAT GCTCCGTGAT GCATGAGGCT CTGCACAACC 1551ACTACACACA GAAGAGCCTC TCCCTGTCTC TGGGTAAA The amino acid sequence of the hingeless C_(H )region of a human IgG4.SEQ ID No: 4:     1ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVG WNSGALTSGV   51HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVAP   101EFLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV   151EVHNAKTKPR EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI   201

  251

  301

The amino acid sequence of the lambda chain constant human(accession number S25751) SEQ ID No: 5:      1qpkaapsvtl fppsseelqa nkatlvclis dfypgavtva wkadsspvka   51gvetttpskg snnkyaassy lsltpeqwks hrsyscgvth egstvektva   101  pteCs The amino acid sequence of the kappa chain constant human(accession number P01834) SEQ ID No: 6:     1tvaapsvfif ppsdeqlksg tasvvcllnn fypreakvqw kvdnalqsgn   51sqesvteqds kdstyslsst ltlskadyek hkvyacevth qglsspvtks   101 fnrgeC The amino acid sequence of IgG1 constant region(accession number P01857) SEQ ID No: 7:     1astkgpsvfp lapSskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv   51

  101

  151 hedpevkfnw yvdgvevhna ktkpreeqyn styrvvsvlt vlhqdwlngk   201

  251

  301

 The amino acid sequence of IgG2 constant region(accession number P01859) SEQ ID No: 8:     1astkgpsvfp lapcsrstse staalgclvk dyfpepvtvs wnsgaltsgv   51

  101

  151 evqfnwyvdg vevhnaktkp reeqfnstfr vvsvltvvhq dwlngkeykc   201

  251

  301

 The amino acid sequence of IgG3 constant region(accession number A23511) SEQ ID No: 9:     1astkgpsvfp lapcsrstsg gtaalgclvk dyfpepvtvs wnsgaltsgv   51

  101

  151

  201 pevqfkwyvd gvevhnaktk preeqynstf rvvsvltvlh qdwlngkeyk   251

  301

  351

1-91. (canceled)
 92. A method of treating a disorder, comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a monovalent antibody comprising a single heavy chain and asingle light chain, wherein the antibody is a human IgG4 isotypecomprising a modified heavy chain constant region, wherein the heavychain constant region has been modified relative to the sequence setforth in SEQ ID NO: 4 by one or more of the following amino acidsubstitutions: Glu (E) in position 225 has been replaced by Ala (A) orVal (V); Ser (S) in position 232 has been replaced by Arg (R); Leu (L)in position 236 has been replaced by Glu (E), Gly (G), Ser (S), or Thr(T); Asp (D) in position 267 has been replaced by Ala (A) or Ser (S);Phe (F) in position 273 has been replaced by Asp (D), Thr (T), Arg (R),Gln (Q), or Tyr (Y); or Tyr (Y) in position 275 has been replaced by Glu(E), Gln (Q), or Lys (K); and wherein the heavy chain constant regionfurther comprises a hinge region modified such that all amino acidresidues in the hinge region which are capable of forming a disulfidebond with an identical constant region in the presence of polyclonalhuman IgG have been deleted or substituted with other amino acidresidues, including modification of all cysteine residues in the hingeregion.
 93. The method of claim 92, wherein the monovalent antibody is ahuman antibody.
 94. The method of claim 92, wherein the monovalentantibody binds to a target selected from erythropoietin, beta-amyloid,thrombopoietin, interferon-alpha (2a and 2b), interferon-beta (1b),interferon-gamma, TNFR I (CD120a), TNFR II (CD120b), IL-1R type 1(CD121a), IL-1R type 2 (CD121b), IL-2, IL-2R (CD25), IL-2R-beta (CD123),IL-3, IL-4, IL-3R (CD123), IL-4R (CD124), IL-5R (CD125), IL-6R-alpha(CD126), IL-6R-beta (CD130), IL-8, IL-10, IL-11, IL-15, IL-15BP, IL-15R,IL-20, IL-21, TCR variable chain, RANK, RANK-L, CTLA4, CXCR4R, CCR5R,TGF-beta1, TGF-beta2, TGF-beta3, G-CSF, GM-CSF, MIF-R (CD74), M-CSF-R(CD115), GM-CSF-R (CD116), soluble FcRI, soluble FcRII, soluble FcRIII,FcRn, Factor VII, Factor VIII, Factor IX, VEGF, VEGFxxxb, alpha-4integrin, CD11 a, CD18, CD20, CD38, CD79, CD81, FcalphaRI, FcepsilonRI,acetylcholine receptor, fas, fasL, TRAIL, hepatitis C virus, envelope E2of hepatitis C virus, tissue factor, a complex of tissue factor andFactor VII, EGFr, CD4, CD28, VLA-1, VLA-2, VLA-3, VLA-4, LFA-1, MAC-1,1-selectin, PSGL-1, ICAM-1, P-selectin, periostin, CD33 (Siglec 3),Siglec 8, TNF, CCL1, CCL2, CCL3, CCL4, CCL5, CCL11, CCL13, CCL17, CCL18,CCL20, CCL22, CCL26, CCL27, CX3CL1, LIGHT, EGF, TGFalpha, HGF, PDGF,NGF, C1q, C4, C2, C3, C5, C6, C7, C8, C9, MBL, factor B, a matrixmetallo protease (MMP), CD32b, CD200, CD200R, a killerimmunoglobulin-like receptor (KIR), NKG2D, a leukocyte-associatedimmunoglobulin-like receptor (LAIR), Iy49, PD-L2, CD26, BST-2, melanomainhibitor of apoptosis protein (ML-IAP), cathepsin D, CD40, CD4OR, CD86,a B cell receptor, c-Met, PD-1, a T cell receptor, erb-B1, erb-B2,erb-B3, erb-B4, ephrin-A1, ephrin-A2, ephrin-A3, ephrin-A4, ephrin-A5,ephrin-A6, ephrin-A7, ephrin-A8, ephrin-B1, ephrin-B2, ephrin-B3,ephrin-B4, ephrin-B5, ephrin-B6, TLR-3, TLR-9, angiopoietin-1,angiopoietin-2, CD30, CD95, an adrenocorticosteroid, insulin, GIP,GLP-1, a thyroid hormone, growth hormone, ACTH, oestrogen, testosterone,anti-diuretic hormone, heparin, EPO, an opiate, morphine, vitamin C, LH,and FSH.
 95. The method of claim 92, wherein the monovalent antibody isconjugated to a therapeutic moiety, an immunosuppressant or aradioisotope.
 96. The method of claim 92, wherein the monovalentantibody comprises all the listed amino acid substitutions relative tothe sequence set forth in SEQ ID NO:
 4. 97. The method of claim 92,wherein the heavy chain constant region of the monovalent antibody hasbeen modified such that all cysteine residues in the hinge region havebeen substituted with amino acid residues that have an uncharged polarside chain or a nonpolar side chain.
 98. The method of claim 92, whereinthe heavy chain constant region of the monovalent antibody has beenmodified relative to the sequence set forth in SEQ ID NO: 4 by thefollowing amino acid substitutions: Phe (F) in position 273 has beenreplaced by Asp (D) or Thr (T), and Tyr (Y) in position 275 has beenreplaced by Glu (E).
 99. The method of claim 92, wherein the heavy chainconstant region of the monovalent antibody has been modified relative tothe sequence set forth in SEQ ID NO: 4 such that Phe (F) in position 273has been replaced by Asp (D).
 100. The method of claim 92, wherein theheavy chain constant region of the monovalent antibody has been modifiedrelative to the sequence set forth in SEQ ID NO: 4 such that Phe (F) inposition 273 has been replaced by Thr (T).
 101. The method of claim 92,wherein the heavy chain constant region of the monovalent antibody hasbeen modified relative to the sequence set forth in SEQ ID NO: 4 suchthat Tyr (Y) in position 275 has been replaced by Glu (E).
 102. Themethod of claim 92, wherein the heavy chain constant region of themonovalent antibody has been modified relative to the sequence set forthin SEQ ID NO: 4 by the following amino acid substitutions: Asp (D) inposition 267 has been replaced by Ser (S), and Tyr (Y) in position 275has been replaced by Glu (E), Gln (Q), or Lys (K).
 103. The method ofclaim 92, wherein the heavy chain constant region of the monovalentantibody has been modified relative to the sequence set forth in SEQ IDNO: 4 such that Asp (D) in position 267 has been replaced by Ser (S).104. The method of claim 92, wherein the heavy chain constant region ofthe monovalent antibody has been modified relative to the sequence setforth in SEQ ID NO: 4 such that Tyr (Y) in position 275 has beenreplaced by Glu (E).
 105. The method of claim 92, wherein the heavychain constant region of the monovalent antibody has been modifiedrelative to the sequence set forth in SEQ ID NO: 4 such that Tyr (Y) inposition 275 has been replaced by Gln (Q).
 106. The method of claim 92,wherein the heavy chain constant region of the monovalent antibody hasbeen modified relative to the sequence set forth in SEQ ID NO: 4 suchthat Tyr (Y) in position 275 has been replaced by Lys (K).
 107. Themethod of claim 92, wherein the monovalent antibody is administered withone or more pharmaceutically acceptable excipients, diluents, and/orcarriers.
 108. The method of claim 92, wherein the monovalent antibodyis administered alone or in combination with one or more additionaltherapeutic agents.
 109. The method of claim 108, wherein the one ormore additional therapeutic agents are selected from one or moreantibodies, one or more cytotoxic agents, one or more radiotoxic agents,one or more anti-angiogenic agents, one or more cytokines, one or moregrowth inhibitory agents, one or more anti-inflammatory agents, one ormore disease modifying anti-rheumatic drugs (DMARDs), and one or moreimmunosuppressants, or any combination thereof.
 110. The method of claim108, wherein the monovalent antibody and the one or more additionaltherapeutic agents are administered simultaneously.
 111. The method ofclaim 110, wherein the monovalent antibody and the one or moreadditional therapeutic agents are in the same formulation.
 112. Themethod of claim 110, wherein the monovalent antibody and the one or moreadditional therapeutic agents are in separate formulations.
 113. Themethod of claim 108, wherein the monovalent antibody and the one or moreadditional therapeutic agents are administered sequentially.
 114. Themethod of claim 113, wherein the monovalent antibody is administeredbefore administration of the one or more additional therapeutic agents,after administration of the one or more additional therapeutic agents,or both.
 115. The method of claim 92, wherein the disorder is selectedfrom a neoplastic disorder or malignancy, an inflammatory or immunedisorder, an angiogenic disorder, an allergic disorder, a neurologicalor neurogenerative disorder, a gastrointestinal disorder, a hepaticdisorder, a hematological disorder, a skin disorder, a pulmonarydisorder, an endocrine disorder, a vascular disorder, an infectiousdisease or disorder, a kidney disorder, an ophthalmologic disorder, acardiac disorder, a circulatory disorder, a metabolic disorder, a bonedisorder, a muscle disorder, a connective tissue disorder, agynecological-obstetrical disorder, a male reproductive disorder, atransplantation-derived disorder, and a viral infection.
 116. The methodof claim 115, wherein the disorder is a neoplastic disorder ormalignancy selected from a leukemia, a lymphoid neoplasm, an ovariancancer, an endometrial cancer, a lung cancer, a brain cancer, a spinalcord cancer, a breast cancer, an oral cancer, a colorectal cancer, apancreatic cancer, a head and neck cancer, a kidney cancer, a thymoma, alung cancer, a skin cancer, a larynx cancer, a liver cancer, a gastriccancer, an esophageal cancer, a prostate cancer, a bladder cancer, asarcoma, a cancer of the testis, and a parotid tumor, or any metastasesthereof.
 117. The method of claim 115, wherein the disorder is selectedfrom B-cell chronic lymphocytic leukemia, acute myeloid leukemia, B-celllymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma,non-cutaneous T-cell lymphoma, non-small cell lung cancer, glioblastoma,glioma, oral squamous cell carcinoma, head and neck squamous cellcarcinoma, melanoma, immune mediated cytopenia, HIV infection, AIDS,cystic fibrosis, osteomyelitis, glomerulonephritis, fibrosis, acuterespiratory distress syndrome, chorioretinitis, palmoplanar pustulosis,alcoholic hepatitis, acute pancreatitis, transplant rejection,graft-versus-host disease, Alzheimer's disease, Parkinson's disease,myocardial vascular disease, cerebral vascular disease, retinopathy,macular degeneration, cardiovascular disease, post-thrombotic vein wallfibrosis, ischemia reperfusion injury, atherosclerosis, stroke, cerebralaneurysm, asthma, allergic rhinitis, hay fever, atopic dermatitis,eczema, hives, urticaria, allergic conjunctivitis, a seasonal allergy, anasal allergy, a contact allergy, an ocular allergy, a food or drugallergy, a latex allergy, an insect allergy, an IgA nephropathy, cornealwound healing, a degenerative genetic eye disease, dental caries,periodontitis, rheumatoid arthritis, gout, multiple sclerosis,inflammatory bowel disease, type-1 diabetes, systemic lupuserythematosus, psoriasis, atopic dermatitis, chronic obstructivepulmonary disease, sepsis, hepatitis C virus infection, Crohn's disease,Guillain-Barre syndrome, ulcerative colitis, hemolytic anemia,paroxysmal nocturnal hemoglobinuria, a heart attack, a burn injury,myasthenia gravis, and anti-phospholipid syndrome.